WO2019192488A1 - 具有靶向性的穿膜肽-多臂聚乙二醇-药物偶联物及其应用 - Google Patents

具有靶向性的穿膜肽-多臂聚乙二醇-药物偶联物及其应用 Download PDF

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WO2019192488A1
WO2019192488A1 PCT/CN2019/081057 CN2019081057W WO2019192488A1 WO 2019192488 A1 WO2019192488 A1 WO 2019192488A1 CN 2019081057 W CN2019081057 W CN 2019081057W WO 2019192488 A1 WO2019192488 A1 WO 2019192488A1
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bond
group
integer
peg
peptide
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PCT/CN2019/081057
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French (fr)
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林美娜
赵宣
朱振刚
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北京键凯科技有限公司
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Priority to KR1020207030356A priority Critical patent/KR20200134287A/ko
Priority to US17/044,866 priority patent/US20220047713A1/en
Publication of WO2019192488A1 publication Critical patent/WO2019192488A1/zh

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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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • 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
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    • 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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to a transmembrane peptide-multi-arm polyethylene glycol-drug conjugate, in particular to a targeted transmembrane peptide-multi-arm polyethylene glycol-drug couple Linkage and its application in biomedicine.
  • CPPs Cellpenetrating peptides
  • CPPs are a class of peptides that enter the cell directly through the cell membrane in a non-receptor-dependent manner. They are generally less than 30 amino acids in length and are rich in basic amino acids. Usually with a positive charge, scientists have applied it to gene therapy. Penetrating the cell membrane into the cell is a prerequisite for many biological macromolecules that act on the cell. However, the biological barrier of the biofilm prevents many high molecular substances from entering the cell, thus greatly limiting these substances. Application in the field of treatment.
  • Non-patent literature “Progress in the study of cell penetrating peptides in drug delivery systems” (Fan Bo et al., J. Pharmaceutica, 2016 (2): 264-271), the currently discovered CPPs mainly include transcriptional transactivators ( Tat), VP22, transportan, membrane type amphiphilic peptide (MAP), signal transduction peptide and arginine-rich sequence peptide.
  • Tat transcriptional transactivators
  • VP22 transportan
  • MAP membrane type amphiphilic peptide
  • signal transduction peptide arginine-rich sequence peptide
  • CPPs can be roughly divided into three categories: cationic peptides, including: R9, TAT, hLF, (RXR) 4, NLSs, AMPs, etc.; amphiphilic peptides, Mainly include: MPG, penetratin, CADY, vascular endothelium-cadherin (pVEC), ARF (1-22), BPrPr (1-28); hydrophobic peptides, mainly including signal sequences found from integrin ⁇ 3 (VTVLALGALAGVGVG And Kaposi fibroblast growth factor (AAVALLPAVLLALLAP).
  • CPPs have strong transport potential, a property that makes it a good carrier for targeted drugs. So far, CPPs have effectively mediated various types of bioactive substances with different molecular weights and particle sizes into cells, such as small molecule drugs, dyes, peptides, peptide nucleic acids, proteins, antibodies, plasmid DNA, small interfering RNA (small interfering). RNA, siRNA), liposomes, phage particles, superparamagnetic particles, fluorescent stains, nanoparticles, viruses, quantum dots, magnetic resonance imaging contrast agents, and the like.
  • CPPs can deliver a variety of substances into cells. Because the physical and chemical properties of the transported substances are different, different connection methods are needed to connect CPPs with the transported substances. Usually, the connection method has an important influence on the intake level and intake mode of CPPs. The connection method is that the covalent bond connection and the electrostatic interaction are connected to each other.
  • the connection method is that the covalent bond connection and the electrostatic interaction are connected to each other.
  • nucleic acid drugs have strong electronegativity, and they will attract each other to form a hairpin structure with positively charged CPPs, so that intracellular delivery cannot be achieved. Does not affect the activity of the drug. This problem can be solved by coupling polyethylene glycol (PEG) soft segments between CPPs and nucleic acid drugs.
  • PEG polyethylene glycol
  • the flexible segments can physically separate nucleic acid drugs from CPPs, thereby preventing strong electronegativity nucleic acid drugs.
  • the positively charged CPPs attract each other to form a hairpin structure, thereby avoiding the aggregation and precipitation of nucleic acid drugs and CPPs due to electrostatic interaction, so that the nucleic acid drugs can exert their biological activities in the cytoplasm or nucleus after entering the cells, thereby improving Its bioavailability.
  • PEG is an extremely versatile polyether polymer compound that can be used in many fields such as medicine, health, food, and chemical industry. PEG can be dissolved in water and many solvents, and the polymer has excellent biocompatibility, can be dissolved in the tissue fluid in the body, and can be quickly excreted by the body without any toxic side effects.
  • the PEGs involved are all linear structures.
  • the patent document CN 105727304 A describes the preparation and application of a class of nucleic acid conjugates, the specific structure of which is a covalently linked transmembrane peptide and a nucleic acid drug respectively at both ends of the flexible segment, and the soft segment is composed of a PEG having a linear structure and a poly Any of oxyethylene, polyoxypropylene, polyethylene, and polyacrylamide is formed.
  • the use of a hydrophilic polymer as a linking group to link CPPs with a nucleic acid drug is described, thereby improving the intracellular drug delivery efficiency
  • the hydrophilic polymer is preferably PEG, which is a linear structure. It can be seen that the existing reports use linear PEG as a linking group to link CPPs with therapeutic drugs, and do not make any improvement on the loading dose in the CPPs forming delivery system, while the linear PEG is separately at its molecular ends. It can be linked to a CPP and a drug molecule with a low CPP or drug connection rate per molecule.
  • the inventors have engineered a linear PEG into a multi-arm PEG having a plurality of terminal groups, having a plurality of functional group introduction points, and capable of linking a plurality of different reactive groups to solve a linear PEG linkage.
  • the problem is limited, the application range is small, and the drug load is low.
  • transmembrane peptides are not selective for cells, can carry drugs into all cells, and have no clear purpose, often leading to pathogenic cells and normal cells. Killing together can not only play a good drug effect with the drug, but also cause certain damage to the body. Therefore, inventing a delivery system that can be targeted to introduce a drug into a disease-causing cell and to exert the maximum utility of the drug is an urgent problem to be solved.
  • a biligand-modified liposome composed of folic acid and the transmembrane peptide PEP-1 can transport anti-tumor drugs into tumor cells by using such a transport system, thereby efficiently and selectively killing tumor cells and reducing Adverse reactions to the drug.
  • the targeting group is attached to the transmembrane peptide with limited binding sites.
  • the inventors designed a transmembrane peptide-multiarm polyethylene glycol-drug conjugate having a targeted and potent cell entry, which not only improved the drug
  • the carrier rate, and the targeting group can be attached to the transmembrane peptide and/or the polyethylene glycol chain and/or the drug molecule, and the drug can target the pathogenic cells to exert maximum therapeutic effect.
  • An object of the present invention is to provide a transmembrane peptide-multi-arm PEG-drug conjugate having multiple end groups having multiple functions compared to a linear PEG-penetrating peptide conjugate.
  • the introduction point of the group can link a plurality of different active groups, and solve the problem that the linear PEG connection site is limited, the application range is small, and the drug load is low.
  • Another object of the invention is to provide the use of a targeted transmembrane peptide-multi-arm PEG-drug conjugate, particularly in the treatment of asthma and pulmonary fibrosis.
  • the R is a central molecule selected from the group consisting of a polyhydroxy structure, a polyamino structure or a polycarboxy structure;
  • the R is selected from the group consisting of: pentaerythritol or polypentaerythritol structure, glycerol or polyglycerol structure, methyl glucoside, sucrose; in a preferred embodiment of the invention, the R is selected from:
  • l is an integer ⁇ 1 and ⁇ 10; preferably, l is an integer ⁇ 1 and ⁇ 10, more preferably, l is an integer ⁇ 1 and ⁇ 7; in a specific embodiment of the invention, the It is preferably 1, 2, 3, 4, 5 or 6.
  • the PEG is the same or different -(CH 2 CH 2 O) m -, the average value of m is an integer from 3 to 250; preferably, the m is an integer from 68 to 250; more preferably, the m An integer of 68-227.
  • the C is a CPP, and is selected from the group consisting of a transcriptional transactivator (Tat), a VP22, a transportan, a membrane-type amphiphilic peptide (MAP), a signal transduction peptide, and an arginine-rich sequence peptide;
  • the C is selected from the group consisting of: LMWP, Tat48-60, Tat48-60-P10, CAI, HIV-TAT, MAP, MPG ⁇ , M918, R6Pen, peneratin, Pep-1-K, ARF1-22, Tp10, POD a polylysine composed of 3-100 lysine residues and a polyarginine composed of 4-9 arginine residues;
  • the C is LMWP and polyarginine consisting of 8 arginine.
  • the D is a drug molecule selected from the group consisting of: a small molecule drug, a dye, a polypeptide, a polypeptide nucleic acid, a protein, an antibody, a plasmid DNA, a nucleic acid, a liposome, a phage particle, a superparamagnetic particle, a fluorescent dye, Nanoparticles, viruses, quantum dots, magnetic resonance imaging contrast agents, in particular siRNA, especially Cytokine-siRNA;
  • the D is selected from a small molecule drug, a polypeptide, an antibody, a nucleic acid
  • the nucleic acid comprises a nucleotide monomer or an oligonucleotide
  • the nucleotide monomer comprises four deoxyribonucleotide monomers and Four lotus pond nucleomonomers
  • the oligonucleotides are a substituted oligonucleotide and a non-substituted oligonucleotide
  • the substituted oligonucleotide is a phosphoryldiamine morpholino oligonucleotide
  • the non-substituted oligonucleotide is selected from the group consisting of a locked nucleic acid, an siRNA, a microRNA, a aptamer, a peptide nucleic acid, a decoy ODN, a catalytic RNA, and a CpG dinucleotide;
  • the D is selected from a monoclonal antibody, and the oligonucleotide is an siRNA having a length of 19 to 23 bp.
  • the D is selected from the group consisting of: omalizumab, nidanib, bevacizumab, pemizumab, trastuzumab, navobizumab, VEGF-siRNA , IL-v-siRNA, Syk-siRNA, GATA-3-siRNA, and v is selected from 4, 5, 8, and 13.
  • the X is a linkage bond of a PEG-linked CPPs, and the linkage is composed of an amide bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a thiol-maleimide bond, a triazole, and a carbon sulfur.
  • j is an integer from 0 to 10; preferably, j is an integer from 0 to 5; more preferably, j is an integer from 0 to 3; in a specific embodiment of the invention, j is 0, 1, 2, 3 , 4 or 5.
  • the Y is a linkage between the PEG and the drug molecule D, and the linkage is selected from the group consisting of: a disulfide bond, a hydrazone bond, an amide bond, an ester bond, an ether bond, a carbonyl bond, a thioester bond, or a thiol-maleyl group.
  • the Y is selected from the group consisting of: a disulfide bond, a hydrazone bond, an amide bond, an ester bond, a thioester bond, and a thiol-maleimide, and the disulfide bond and the hydrazone bond are used for cytoplasmic release;
  • the amide bond, ester bond, thioester bond and mercapto-maleimide are used for intranuclear drug release.
  • n is the number of branches or the number of arms, an integer of n ⁇ 3; preferably, the n is an integer from 3 to 22; more preferably, the n is an integer from 3 to 14, most preferably, the n Is a 3-8 integer.
  • the k is the number of branches or the number of arms connecting the CPPs, 1 ⁇ k ⁇ n; preferably, the k is an integer from 1 to 14; more preferably, the k is an integer from 1 to 6; in the present invention In an embodiment, the k is 1, 2, 3, 6.
  • the transmembrane peptide-multiarm PEG-drug conjugate of the formula I is structured as follows:
  • R, PEG, C, X, Y, n, k are as defined in the present invention.
  • the D is a drug molecule selected from the group consisting of: a small molecule drug, a dye, a polypeptide, a polypeptide nucleic acid, a protein, an antibody, a plasmid DNA, a nucleic acid, a liposome, a phage particle, a superparamagnetic particle, a fluorescent dye, Nanoparticles, viruses, quantum dots, magnetic resonance imaging contrast agents, in particular siRNA, especially Cytokine-siRNA;
  • the D is selected from a small molecule drug, a polypeptide, an antibody, a nucleic acid
  • the nucleic acid comprises a nucleotide monomer or an oligonucleotide
  • the nucleotide monomer comprises four deoxyribonucleotide monomers and Four lotus pond nucleomonomers
  • the oligonucleotides are a substituted oligonucleotide and a non-substituted oligonucleotide
  • the substituted oligonucleotide is a phosphoryldiamine morpholino oligonucleotide
  • the non-substituted oligonucleotide is selected from the group consisting of a locked nucleic acid, an siRNA, a microRNA, a aptamer, a peptide nucleic acid, a decoy ODN, a catalytic RNA, and a CpG dinucleotide;
  • said D is selected from the group consisting of siRNAs having an oligonucleotide length of 19-23 bp.
  • the T is a targeting group, and the T is selected from the group consisting of: a protein, an antibody, an antibody fragment or a derivative thereof, a small molecule peptide, a polypeptide, glucose, galactose, folic acid, hyaluronic acid;
  • the antibody is a monoclonal antibody, and the antibody fragment or derivative thereof is a single chain of an Fv or Fab fragment.
  • the T is selected from the group consisting of folic acid, RGD, cRGD, hyaluronic acid, glucose, galactose.
  • the B is a transmembrane peptide or a linkage bond between a drug molecule and a targeting group, and the linkage is composed of an amide bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a thiol-maleimide bond, One or more of a carbon-sulfur bond or an ether bond;
  • the B is selected from the group consisting of -(CH 2 ) j CONH(CH 2 ) j -, -(CH 2 ) j -SS-(CH 2 ) j -, -(CH 2 ) j COO(CH 2 ) j
  • -, -(CH 2 ) j NH-N C(CH 2 ) j -, -(CH 2 ) j -S-(CH 2 ) j - ;
  • j is an integer from 0 to 10; preferably, j is an integer from 0 to 5; more preferably, j is an integer from 0 to 3; in a specific embodiment of the invention, j is 0, 1, 2, 3 , 4 or 5.
  • the transmembrane peptide-multiarm PEG-drug conjugate of the formula II or III has the following structure:
  • the R is a central molecule selected from the group consisting of a polyhydroxy structure, a polyamino structure or a polycarboxy structure;
  • the R is selected from the group consisting of: pentaerythritol or polypentaerythritol structure, glycerol or polyglycerol structure, methyl glucoside, sucrose; in a preferred embodiment of the invention, the R is selected from:
  • l is an integer ⁇ 1 and ⁇ 10; preferably, l is an integer ⁇ 1 and ⁇ 10, more preferably, l is an integer ⁇ 1 and ⁇ 7; in a specific embodiment of the invention, the It is preferably 1, 2, 3, 4, 5 or 6.
  • the PEG is the same or different -(CH 2 CH 2 O) m -, the average value of m is an integer from 3 to 250; preferably, the m is an integer from 68 to 250; more preferably, the m An integer of 68-227.
  • the C is a CPP, and is selected from the group consisting of a transcriptional transactivator (Tat), a VP22, a transportan, a membrane-type amphiphilic peptide (MAP), a signal transduction peptide, and an arginine-rich sequence peptide;
  • the C is selected from the group consisting of: LMWP, Tat48-60, Tat48-60-P10, CAI, HIV-TAT, MAP, MPG ⁇ , M918, R6Pen, penetratin, Pep-1-K, ARF1-22, Tp10, POD a polylysine composed of 3-100 lysine residues and a polyarginine composed of 4-9 arginine residues;
  • the C is LMWP and polyarginine consisting of 8 arginine.
  • the D is a drug molecule selected from the group consisting of: a small molecule drug, a dye, a polypeptide, a polypeptide nucleic acid, a protein, an antibody, a plasmid DNA, a nucleic acid, a liposome, a phage particle, a superparamagnetic particle, a fluorescent dye, Nanoparticles, viruses, quantum dots, magnetic resonance imaging contrast agents, in particular siRNA, especially Cytokine-siRNA;
  • the D is selected from a small molecule drug, a polypeptide, an antibody, a nucleic acid
  • the nucleic acid comprises a nucleotide monomer or an oligonucleotide
  • the nucleotide monomer comprises four deoxyribonucleotide monomers and Four lotus pond nucleomonomers
  • the oligonucleotides are a substituted oligonucleotide and a non-substituted oligonucleotide
  • the substituted oligonucleotide is a phosphoryldiamine morpholino oligonucleotide
  • the non-substituted oligonucleotide is selected from the group consisting of a locked nucleic acid, an siRNA, a microRNA, a aptamer, a peptide nucleic acid, a decoy ODN, a catalytic RNA, and a CpG dinucleotide;
  • the D is selected from a monoclonal antibody, and the oligonucleotide is an siRNA having a length of 19 to 23 bp.
  • the T is a targeting group, and the T is selected from the group consisting of: a protein, an antibody, an antibody fragment or a derivative thereof, a small molecule peptide, a polypeptide, glucose, galactose, folic acid, hyaluronic acid;
  • the antibody is a monoclonal antibody, and the antibody fragment or derivative thereof is a single chain of an Fv or Fab fragment.
  • the T is selected from the group consisting of folic acid, RGD, cRGD, hyaluronic acid, glucose, galactose.
  • the X is a linkage bond of a PEG-linked CPPs, and the linkage is composed of an amide bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a thiol-maleimide bond, a triazole, and a carbon sulfur.
  • the X is selected from the group consisting of -(CH 2 ) j CONH(CH 2 ) j -, -(CH 2 ) j -SS-(CH 2 ) j -, -(CH 2 ) j COO(CH 2 ) j
  • -, -(CH 2 ) j NH-N C(CH 2 ) j -, -(CH 2 ) j -S-(CH 2 ) j - ;
  • j is an integer from 0 to 10; preferably, j is an integer from 0 to 5; more preferably, j is an integer from 0 to 3; in a specific embodiment of the invention, j is 0, 1, 2, 3 , 4 or 5.
  • the Y is a linkage between the PEG and the drug molecule D, and the linkage is selected from the group consisting of: a disulfide bond, a hydrazone bond, an amide bond, an ester bond, an ether bond, a carbonyl bond, a thioester bond, or a thiol-maleyl group.
  • the Y is selected from the group consisting of: a disulfide bond, a hydrazone bond, an amide bond, an ester bond, a thioester bond, and a thiol-maleimide, and the disulfide bond and the hydrazone bond are used for cytoplasmic release;
  • the amide bond, ester bond, thioester bond and mercapto-maleimide are used for intranuclear drug release.
  • the B is a linkage between a PEG and a targeting group, and the linkage is an amide bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a thiol-maleimide bond, a carbon sulfur bond or an ether.
  • the keys are an amide bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a thiol-maleimide bond, a carbon sulfur bond or an ether.
  • the B is selected from the group consisting of -(CH 2 ) j CONH(CH 2 ) j -, -(CH 2 ) j -SS-(CH 2 ) j -, -(CH 2 ) j COO(CH 2 ) j
  • -, -(CH 2 ) j NH-N C(CH 2 ) j -, -(CH 2 ) j -S-(CH 2 ) j - ;
  • j is an integer from 0 to 10; preferably, j is an integer from 0 to 5; more preferably, j is an integer from 0 to 3; in a specific embodiment of the invention, j is 0, 1, 2, 3 , 4 or 5.
  • n is the number of branches or the number of arms, an integer of n ⁇ 3; preferably, the n is an integer from 3 to 22; more preferably, the n is an integer from 3 to 14, most preferably, the n Is a 3-8 integer.
  • the k is the number of branches or the number of arms connecting the CPPs, 1 ⁇ k ⁇ n; preferably, the k is an integer from 1 to 14; more preferably, the k is an integer from 1 to 6; in the present invention In an embodiment, the k is 1, 2, 4, 6.
  • the g is the number of branches or the number of arms to which the targeting group is attached, 1 ⁇ g ⁇ n; preferably, the g is an integer from 1 to 8; more preferably, the g is an integer from 1 to 4; In an embodiment of the invention, the g is 1, 2, 3, 4.
  • transmembrane peptide-multiarm PEG-drug conjugate of the formula IV is as follows:
  • the R is a central molecule selected from the group consisting of a polyhydroxy structure, a polyamino structure or a polycarboxy structure;
  • the R is selected from the group consisting of: pentaerythritol or polypentaerythritol structure, glycerol or polyglycerol structure, methyl glucoside, sucrose; in a preferred embodiment of the invention, the R is selected from:
  • l is an integer ⁇ 1 and ⁇ 10; preferably, l is an integer ⁇ 1 and ⁇ 10, more preferably, l is an integer ⁇ 1 and ⁇ 7; in a specific embodiment of the invention, the It is preferably 1, 2, 3, 4, 5 or 6.
  • the PEG is the same or different -(CH 2 CH 2 O) m -, the average value of m is an integer from 3 to 250; preferably, the m is an integer from 68 to 250; more preferably, the m An integer of 68-227.
  • the C is a CPP, and is selected from the group consisting of a transcriptional transactivator (Tat), a VP22, a transportan, a membrane-type amphiphilic peptide (MAP), a signal transduction peptide, and an arginine-rich sequence peptide;
  • the C is selected from the group consisting of: LMWP, Tat48-60, Tat48-60-P10, CAI, HIV-TAT, MAP, MPG ⁇ , M918, R6Pen, peneratin, Pep-1-K, ARF1-22, Tp10, POD a polylysine composed of 3-100 lysine residues and a polyarginine composed of 4-9 arginine residues;
  • the C is LMWP and polyarginine consisting of 8 arginine.
  • the D and D' are independently selected from the group consisting of: small molecule drugs, dyes, polypeptides, polypeptide nucleic acids, proteins, antibodies, plasmid DNA, nucleic acids, liposomes, phage particles, superparamagnetic particles, fluorescent dyes, nanoparticles , viruses, quantum dots, magnetic resonance imaging contrast agents, especially siRNAs, especially Cytokine-siRNA;
  • the D is selected from a small molecule drug; more preferably, the D is selected from a small molecule drug having a molecular weight of less than 1000, such as vitamin C, acetylsalicylic acid, acetaminophen, acetamino group Phenol and the like.
  • the D' is selected from a nucleic acid comprising a nucleotide monomer or an oligonucleotide;
  • the nucleotide monomer comprises four deoxyribonucleotide monomers and four lotus pond nucleotides
  • Oligonucleotides are substituted oligonucleotides and non-substituted oligonucleotides, and the substituted oligonucleotides are phosphoryl diamine morpholino oligonucleotides, and the non-substituted oligonucleotides are selected.
  • the D' is selected from the group consisting of a monoclonal antibody, and the oligonucleotide is an siRNA having a length of 19 to 23 bp.
  • the X is a linkage bond of a PEG-linked CPPs, and the linkage is composed of an amide bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a thiol-maleimide bond, a triazole, and a carbon sulfur.
  • the X is selected from the group consisting of -(CH 2 ) j CONH(CH 2 ) j -, -(CH 2 ) j -SS-(CH 2 ) j -, -(CH 2 ) j COO(CH 2 ) j
  • -, -(CH 2 ) j NH-N C(CH 2 ) j -, -(CH 2 ) j -S-(CH 2 ) j - ;
  • j is an integer from 0 to 10; preferably, j is an integer from 0 to 5; more preferably, j is an integer from 0 to 3; in a specific embodiment of the invention, j is 0, 1, 2, 3 , 4 or 5.
  • the Y is a linkage between the PEG and the drug molecule D, and the linkage is selected from the group consisting of: a disulfide bond, a hydrazone bond, an amide bond, an ester bond, an ether bond, a carbonyl bond, a thioester bond, or a thiol-maleyl group.
  • the Y is selected from the group consisting of: a disulfide bond, a hydrazone bond, an amide bond, an ester bond, a thioester bond, and a thiol-maleimide, and the disulfide bond and the hydrazone bond are used for cytoplasmic release;
  • the amide bond, ester bond, thioester bond and mercapto-maleimide are used for intranuclear drug release.
  • the Z is a linkage between the PEG and the drug molecule D', and the linkage is composed of an amide bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a thiol-maleimide bond, a carbon-sulfur bond. Or one or more of the ether bonds;
  • the Z is selected from the group consisting of -(CH 2 ) j CONH(CH 2 ) j -, -(CH 2 ) j -SS-(CH 2 ) j -, -(CH 2 ) j COO(CH 2 ) j
  • -, -(CH 2 ) j NH-N C(CH 2 ) j -, -(CH 2 ) j -S-(CH 2 ) j - ;
  • j is an integer from 0 to 10; preferably, j is an integer from 0 to 5; more preferably, j is an integer from 0 to 3; in a specific embodiment of the invention, j is 0, 1, 2, 3 , 4 or 5.
  • n is the number of branches or the number of arms, an integer of n ⁇ 3; preferably, the n is an integer from 3 to 22; more preferably, the n is an integer from 3 to 14, most preferably, the n Is a 3-8 integer.
  • the k is the number of branches or the number of arms connecting the CPPs, 1 ⁇ k ⁇ n; preferably, the k is an integer from 1 to 14; more preferably, the k is an integer from 1 to 6; in the present invention In an embodiment, the k is 1, 2, 4, 6.
  • the p is the number of branches or the number of arms connecting the drug molecule D', 1 ⁇ p ⁇ n; preferably, the p is an integer from 1 to 8; more preferably, the p is an integer from 1 to 4; In an embodiment of the invention, the p is 1, 2, 3, 4.
  • the transmembrane peptide-multiarm PEG-drug conjugate of the formula V is structured as follows:
  • the PEG has a molecular weight of from 1000 to 80,000 Da; preferably, the PEG has a molecular weight of from 3,000 to 20,000 Da; more preferably, the PEG has a molecular weight of from 3,000 to 10,000 Da; in a most preferred embodiment of the invention
  • the PEG molecular weight may be 3000 Da, 5000 Da, 10000 Da, 20000 Da.
  • the invention also provides a pharmaceutical composition comprising a transmembrane peptide-multiarm PEG-drug conjugate of the formula I, II, III, IV or V.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients selected from the group consisting of carriers, diluents, binders, lubricants, wetting agents.
  • pharmaceutically acceptable excipients selected from the group consisting of carriers, diluents, binders, lubricants, wetting agents.
  • the pharmaceutical composition comprises: a tablet, a capsule, a pill, an injection, an emulsion, a microemulsion, a nanoparticle, an inhalant, a tablet, a gel, a powder, a suppository, a hang emulsion, a cream, and a gel. Freezing agent, spray.
  • the pharmaceutical composition can be administered by oral, subcutaneous, intramuscular, intravenous, rectal, vaginal, nasal, transdermal, subconjunctival, eyeballs.
  • Internal administration eyelid administration, post-ocular administration, retinal administration, choroidal administration, intrathecal injection.
  • transmembrane peptide-multiarm PEG-drug conjugate of the formula I, II, III, IV and V for the preparation of a targeted medicament for the diagnosis and treatment of a disease;
  • the disease is selected from the group consisting of tumor, pneumonia, asthma, pulmonary fibrosis, viral infection, hepatitis, ocular macular degeneration, etc.; preferably, the disease is selected from the group consisting of ocular macular degeneration, asthma, pulmonary fibrosis .
  • the transmembrane peptide-multi-arm PEG-drug conjugates of the general formula I, II, III/IV and V provided by the present invention have a multi-arm PEG compared to a linear PEG-penetrating peptide conjugate.
  • a plurality of end groups having multiple functional groups can be connected to a plurality of different reactive groups, thereby avoiding the problem of limited linear PEG attachment sites, small application range, and low drug loading.
  • the transmembrane peptide-multi-arm PEG-drug conjugate provided by the invention has the targeting property, and the targeting group can be attached to the transmembrane peptide, the drug molecule, or the PEG chain end according to specific therapeutic needs. Targeting drugs into pathogenic cells for precise therapeutic purposes.
  • amino acid sequences of the CPPs of the present invention are as follows:
  • the LMWP was dissolved in 20 mM KH2PO4 buffer solution, 4arm PEG-1arm NHS-3arm Opss was dissolved in DMSO, then added dropwise to the buffer solution, reacted at room temperature for 2 hours, filtered, and purified by heparin column to obtain the product by freeze drying. Or precipitation method;
  • the 4arm PEG-1arm LMWP-MAL-3arm OPSS obtained above was dissolved in dichloromethane, and an appropriate amount of RGD was added thereto, and the reaction was carried out for 12 hours at room temperature, and the reaction liquid was concentrated, purified, freeze-dried or precipitated with isopropyl alcohol. ;
  • sequences of the sense and antisense strands of SH-VEGF-siRNA are as follows:
  • Antisense chain 3'-UUCUAUCUCGUUCUGUUCUUU-5'
  • the gel electrophoresis shows that the product is a strip.
  • the sample was desalted with an ultrafiltration centrifuge tube having a molecular weight cut off of 3000, and the product was obtained by freeze-drying or precipitation, and the product was stored at -20 ° C.
  • the LMWP was dissolved in a 20 mM KH2PO4 buffer solution, 4arm PEG-2arm NHS-2arm Opss was dissolved in DMSO, then added dropwise to a buffer solution, reacted at room temperature for 2 hours, filtered, and purified by heparin column to obtain a product by freeze drying. In the manner obtained, the next reaction can also be directly carried out with the solution;
  • the gel electrophoresis shows that the product is a strip.
  • the sample was desalted with an ultrafiltration centrifuge tube having a molecular weight cut off of 3000, and the product was obtained by freeze-drying or precipitation, and the product was stored at -20 ° C.
  • the LMWP was dissolved in 20 mM KH2PO4 buffer solution, 4arm PEG-3arm NHS-1arm Opss was dissolved in DMSO, then added dropwise to the buffer solution, reacted at room temperature for 2 hours, filtered, and purified by heparin column to obtain the product by freeze drying. In the manner obtained, the next reaction can also be directly carried out with the solution;
  • the gel electrophoresis shows that the product is a strip.
  • the sample was desalted with an ultrafiltration centrifuge tube having a molecular weight cut off of 3000, and the product was obtained by freeze-drying or precipitation, and the product was stored at -20 ° C.
  • the LMWP was dissolved in a 20 mM KH2PO4 buffer solution, 8arm PEG-1arm NHS-7arm Opss was dissolved in DMSO, then added dropwise to a buffer solution, reacted at room temperature for 2 hours, filtered, and purified by heparin column to obtain a product by freeze drying. Or precipitation method;
  • the gel electrophoresis shows that the product is a strip.
  • the sample was desalted with an ultrafiltration centrifuge tube having a molecular weight cut off of 3000, and the product was obtained by freeze-drying or precipitation, and the product was stored at -20 ° C.
  • the LMWP was dissolved in 20 mM KH2PO4 buffer solution, 8arm PEG-2arm NHS-6arm Opss was dissolved in DMSO, then added dropwise to the buffer solution, reacted at room temperature for 2 hours, filtered, and purified by heparin column to obtain the product by freeze drying. Or precipitation method;
  • the gel electrophoresis shows that the product is a strip.
  • the sample was desalted with an ultrafiltration centrifuge tube having a molecular weight cut off of 3000, and the product was obtained by freeze-drying or precipitation, and the product was stored at -20 ° C.
  • the LMWP-PEG5000-VEGF-siRNA was transfected with the four-arm PEG-LMWP-(VEGF-siRNA) 3 (MW 10000) prepared in Example 5 on 293T cells, and the expression of VEGFA in the samples was detected by RT-PCR.
  • the cell effect of the four-arm PEG-LMWP-(VEGF-siRNA) 3 (MW 10000) was significantly better than that of the LMWP-PEG5000-VEGF-siRNA.

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Abstract

本发明提供通式为Ⅰ、Ⅱ、Ⅲ、Ⅳ或Ⅴ的穿膜肽-多臂PEG-药物偶联物，其与直链型PEG-穿膜肽偶联物相比，多臂PEG具有多个端基，有多个功能基团的引入点，可以连接多个不同的活性基团，避免了直链型PEG连接位点有限，应用范围小、药物负载量低的问题。另外，本发明所提供的穿膜肽-多臂PEG-药物偶联物具有靶向性，可根据具体治疗需要，在穿膜肽上、药物分子上、或PEG链端连接靶向基团，使药物靶向地进入致病细胞，达到精准治疗目的。本发明还提供一种具有靶向性的穿膜肽-多臂PEG-药物偶联物的在制备靶向药物中的应用，特别是在治疗眼部黄斑性病变、哮喘和肺部纤维化中的应用。

Description

[根据细则37.2由ISA制定的发明名称]　具有靶向性的穿膜肽-多臂聚乙二醇-药物偶联物及其应用 技术领域
本发明属于生物医药领域，具体涉及一种穿膜肽-多臂聚乙二醇-药物偶联物，尤其是涉及一种具有靶向性的穿膜肽-多臂聚乙二醇-药物偶联物及其在生物医药方面的应用。
背景技术
穿膜肽(cellpenetrating peptides，CPPs)是一类以非受体依赖方式，非经典内吞方式直接穿过细胞膜进入细胞的多肽，其长度一般不超过30个氨基酸且富含碱性氨基酸，氨基酸序列通常带正电荷，目前已有科学家将其应用于基因治疗。穿透细胞膜进入细胞内是许多作用靶点在细胞内的生物大分子发挥作用的先决条件，然而生物膜的生物屏障作用阻止了许多高分子物质进入细胞内，从而很大程度地限制了这些物质在治疗领域的应用。
非专利文献“细胞穿膜肽在药物递送系统中的研究进展”(范博等，药学学报，2016(2):264-271)中描述，目前已发现的CPPs主要包括转录反式激活蛋白(Tat)、VP22、transportan、膜型两亲性肽(MAP)、信号转导肽和富含精氨酸序列肽。根据氨基酸序列、疏水性和极性等方面的特征，CPPs大致可以分为3类：阳离子型肽，主要包括：R9、TAT、hLF、(RXR)4、NLSs、AMPs等；两亲性肽，主要包括：MPG、penetratin、CADY、血管内皮-钙黏蛋白(pVEC)、ARF(1-22)、BPrPr(1-28)；疏水性肽，主要包括从整合素β3中发现的信号序列(VTVLALGALAGVGVG)以及卡波济成纤维细胞生长因子(AAVALLPAVLLALLAP)。
CPPs具有强大的运输潜能，这一性质为其成为靶向药物的良好载体提供了可能。迄今为止，CPPs已经有效地介导了不同分子量和粒径的各类具有生物活性物质进入细胞，如小分子药物、染料、多肽、多肽核酸、蛋白质、抗体、质粒DNA、小干扰RNA(small interfering RNA，siRNA)、脂质体、噬菌体颗粒、超顺磁性粒子、荧光染剂、纳米粒、病毒、量子点、磁共振成像造影剂等。
CPPs能够递送多种类物质进入细胞，由于被转运物质的理化性质不尽相同，需要不同的连接方式将CPPs与被转运物质连接，通常连接方式对CPPs的摄取水平和摄取方式 有重要影响，常用的连接方式为共价键连接和静电作用相互连接。目前关于CPPs介导的核酸类药物递送的研究较多，从研究来看，核酸药物电负性较强，会与呈正电性CPPs之间相互吸引形成发夹结构，从而无法实现胞内递送且不影响药物活性。通过在CPPs与核酸类药物之间偶联聚乙二醇(PEG)柔性链段能够解决这个问题，柔性链段能够在物理空间上间隔核酸药物与CPPs，进而阻止强电负性的核酸药物与呈正电性CPPs之间相互吸引形成发夹结构，从而避免了核酸药物与CPPs间因静电作用而发生的聚集沉淀作用，使得核酸类药物进入细胞后能够在细胞质或细胞核内发挥其生物活性，提高其生物利用度。
PEG是一种用途极为广泛的聚醚高分子化合物，它可应用于医药、卫生、食品、化工等众多领域。PEG能够溶解于水和许多溶剂中，且该聚合物具有优异的生物相容性，在体内能溶于组织液中，能被机体迅速排出体外而不产生任何毒副作用。
现有技术关于CPPs连接PEG作为药物递送系统中，所涉及PEG均为直链型结构。如专利文献CN 105727304 A介绍了一类核酸偶联物制备及其应用，其具体结构为柔性链段两端分别共价连接穿膜肽和核酸药物，柔性链段由具有线性结构的PEG、聚氧乙烯、聚氧丙烯、聚乙烯及聚丙烯酰胺中任意一种形成。又如专利文献EP 1797901 A1和US2013137644 A1描述了利用亲水聚合物作为连接基团连接CPPs与核酸药物，以此提高药物细胞内递送效率，其亲水聚合物优选PEG，为直链型结构。可见，已有的报道均是利用直链型PEG作为连接基团连接CPPs与治疗药物，且未对CPPs形成递送系统中负载药量做任何改进，而直链型PEG是在其分子两端分别可连接一个CPPs和一个药物分子，每个分子中CPPs或药物连接率较低。在本发明中，发明人将直链PEG改造为多臂PEG，其具有多个端基，有多个功能基团的引入点，可以连接多个不同的活性基团，解决直链型PEG连接位点有限，应用范围小、药物负载量低的问题。
在穿膜肽的实际应用中研究者发现了另一个问题，就是穿膜肽对细胞没有选择性，能够携带药物进入所有的细胞，没有明确的目的性，常常会出现将致病细胞和正常细胞一并杀死，既发挥不了所载带药物的良好药效，还会对机体带来一定的损害。因此发明一种可以靶向的将药物导入致病细胞且能够发挥药物最大效用的递送体系是丞待解决的问题。非 专利文献“穿膜肽在肿瘤靶向治疗中的应用，张丽等，肿瘤防治研究，2015,42(10):1043-1048.”中提到穿膜肽在肿瘤靶向治疗中的应用主要有三方面：1，利用酸敏感穿膜肽修饰抗肿瘤药物提高靶向性；2，利用靶向穿膜肽修饰抗肿瘤药物提高抗肿瘤药物的选择性；3，穿膜肽基于肿瘤细胞表面特异性受体实现靶向转运。例如：将叶酸和穿膜肽PEP-1构成的双配体修饰脂质体，利用此种转运系统，就能将抗肿瘤药物转运到肿瘤细胞中，高效、选择性的杀死肿瘤细胞，降低药物的不良反应。但是，仅将靶向基团连接到穿膜肽上，连接位点有限。
为了改善现有技术的缺陷，在本发明中，发明人设计了一种具有靶向性并可强效入胞的穿膜肽-多臂聚乙二醇-药物偶联物，不仅提高了药物载带率，而且靶向基团可连接到穿膜肽上和/或聚乙二醇链上和/或药物分子上，药物可以靶向地进入致病细胞发挥最大治疗效果。
发明内容
本发明一个目的是提供的一种穿膜肽-多臂PEG-药物偶联物，与直链型PEG-穿膜肽偶联物相比，多臂PEG具有多个端基，有多个功能基团的引入点，可以连接多个不同的活性基团，解决直链型PEG连接位点有限，应用范围小、药物负载量低的问题。
本发明还一个目的是提供一种具有靶向性的穿膜肽-多臂PEG-药物偶联物，可根据具体治疗需要，在穿膜肽或药物分子上连接靶向基团，使药物靶向地进入致病细胞，达到精准治疗目的。
本发明还一个目的是提供一种具有靶向性的穿膜肽-多臂PEG-药物偶联物，可根据具体治疗需要，在多臂PEG的链端连接靶向基团，使药物靶向地进入致病细胞，使药物发挥最大疗效。
本发明另一个目的是提供一种具有靶向性的穿膜肽-多臂PEG-药物偶联物的应用，特别是在治疗哮喘和肺部纤维化中的应用。
一种通式为Ⅰ的穿膜肽-多臂PEG-药物偶联物：
Figure PCTCN2019081057-appb-000001
所述R为中心分子，选自：多羟基结构、多氨基结构或多羧基结构；
优选的，所述R选自：季戊四醇或聚季戊四醇结构、丙三醇或聚丙三醇结构、甲基葡萄糖甙、蔗糖；在本发明的优选实施方式中，所述R选自：
Figure PCTCN2019081057-appb-000002
其中l为≥1且≤10的整数；优选的，l为≥1且≤10的整数，更优选，l为≥1且≤7的整数；在本发明的具体实施方式中，所述的l优选为1、2、3、4、5或6。
所述PEG为相同或不同的-(CH 2CH 2O) m-，m的平均值为3-250的整数；优选的，所述m为68-250的整数；更优选的，所述m为68-227的整数。
所述C为CPPs，选自转录反式激活蛋白(Tat)、VP22、transportan、膜型两亲性肽(MAP)、信号转导肽和富含精氨酸序列肽；
优选的，所述C选自：LMWP、Tat48-60、Tat48-60-P10、CAI、HIV-TAT、MAP、MPGα、M918、R6Pen、penetratin、Pep-1-K、ARF1-22、Tp10、POD、3-100个赖氨酸残基组成的聚赖氨酸、4-9个精氨酸残基组成的聚精氨酸；
更优选的，所述C为LMWP和由8个精氨酸组成的聚精氨酸。
所述D为药物分子，所述药物分子选自：小分子药物、染料、多肽、多肽核酸、蛋白质、抗体、质粒DNA、核酸、脂质体、噬菌体颗粒、超顺磁性粒子、荧光染剂、纳米粒、病毒、量子点、磁共振成像造影剂，特别是siRNA，尤其是Cytokine-siRNA；
优选的，所述D选自小分子药物、多肽、抗体、核酸，所述核酸包括核苷酸单体或寡聚核苷酸；核苷酸单体包括四种脱氧核糖核苷酸单体和四种荷塘核苷酸单体；寡聚核苷酸为取代寡核苷酸和非取代寡核苷酸，所述取代寡核苷酸为磷酰二胺吗啉代寡核苷酸，所述 非取代寡核苷酸选自锁核酸、siRNA、microRNA、核酸适体、肽核酸、诱骗ODN、催化性RNA以及CpG二核苷酸中的任意一种；
更优选的，所述D选自单克隆抗体、寡聚核苷酸为长度为19-23bp的siRNA。
在本发明的优选实施方式中，所述D选自：奥马珠单抗、尼达尼布、贝伐珠单抗、派姆单抗、曲妥珠单抗、纳武单抗、VEGF-siRNA、IL-v-siRNA、Syk-siRNA、GATA-3-siRNA，v选自4，5，8，13。
所述X为PEG连接CPPs的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、-三氮唑-、碳硫键或醚键中的一种或两种以上组成；
优选的，所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-、-三氮唑-、巯基-马来酰亚胺键中的一种或两种以上组合；
更优选的，所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-、-三氮唑-、巯基-马来酰亚胺键；
其中j为0-10的整数；优选的，j为0-5的整数；更优选的，j为0-3的整数；在本发明的具体实施方式中，j为0、1、2、3、4或5。
所述Y为PEG与药物分子D之间的连接键，所述连接键选自：二硫键、腙键、酰胺键、酯键、醚键、羰基键、硫酯键或巯基-马来酰亚胺；
优选的，所述Y选自：二硫键、腙键、酰胺键、酯键、硫酯键和巯基-马来酰亚胺，所述的二硫键和腙键用于细胞质释药；所述的酰胺键、酯键、硫酯键和巯基-马来酰亚胺用于细胞核内释药。
所述n为分支数或臂数，n≥3的整数；优选的，所述n为3-22的整数；更优选的，所述n为3-14的整数，最优选的，所述n为3-8整数。
所述k为连接CPPs端的分支数或臂数，1≤k≤n；优选的，所述k为1-14的整数；更优选的，所述k为1-6的整数；在本发明的实施方式中，所述k为1、2、3、6。
在本发明的具体实施方式中，所述的通式为Ⅰ的穿膜肽-多臂PEG-药物偶联物结构如下：
Figure PCTCN2019081057-appb-000003
一种通式为Ⅱ或Ⅲ的穿膜肽-多臂PEG-药物偶联物：
Figure PCTCN2019081057-appb-000004
所述的R、PEG、C、X、Y、n、k的含义如本发明所述定义。
所述D为药物分子，所述药物分子选自：小分子药物、染料、多肽、多肽核酸、蛋白质、抗体、质粒DNA、核酸、脂质体、噬菌体颗粒、超顺磁性粒子、荧光染剂、纳米粒、病毒、量子点、磁共振成像造影剂，特别是siRNA，尤其是Cytokine-siRNA；
优选的，所述D选自小分子药物、多肽、抗体、核酸，所述核酸包括核苷酸单体或寡聚核苷酸；核苷酸单体包括四种脱氧核糖核苷酸单体和四种荷塘核苷酸单体；寡聚核苷酸为取代寡核苷酸和非取代寡核苷酸，所述取代寡核苷酸为磷酰二胺吗啉代寡核苷酸，所述非取代寡核苷酸选自锁核酸、siRNA、microRNA、核酸适体、肽核酸、诱骗ODN、催化性RNA以及CpG二核苷酸中的任意一种；
更优选的，所述D选自寡聚核苷酸长度为19-23bp的siRNA。
所述T为靶向基团，所述T选自：蛋白质、抗体、抗体片段或其衍生物、小分子肽、多肽、葡萄糖、半乳糖、叶酸、透明质酸；
优选的，所述抗体为单克隆抗体，所述抗体片段或其衍生物是Fv或Fab片段的单链。
在本发明的优选实施方式中，所述T选自：叶酸、RGD、cRGD、透明质酸、葡萄糖、半乳糖。
所述B为穿膜肽或药物分子与靶向基团的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、碳硫键或醚键中的一种或两种以上组成；
优选的，所述B选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) j-S-(CH 2) j-中的一种或两种以上组合；
更优选的，所述B选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) j-S-(CH 2) j-；
其中j为0-10的整数；优选的，j为0-5的整数；更优选的，j为0-3的整数；在本发明的具体实施方式中，j为0、1、2、3、4或5。
在本发明的具体实施方式中，所述通式为Ⅱ或Ⅲ的穿膜肽-多臂PEG-药物偶联物结构如下：
Figure PCTCN2019081057-appb-000005
Figure PCTCN2019081057-appb-000006
一种通式为Ⅳ的穿膜肽-多臂PEG-药物偶联物：
Figure PCTCN2019081057-appb-000007
所述R为中心分子，选自：多羟基结构、多氨基结构或多羧基结构；
优选的，所述R选自：季戊四醇或聚季戊四醇结构、丙三醇或聚丙三醇结构、甲基葡萄糖甙、蔗糖；在本发明的优选实施方式中，所述R选自：
Figure PCTCN2019081057-appb-000008
其中l为≥1且≤10的整数；优选的，l为≥1且≤10的整数，更优选，l为≥1且≤7的整数；在本发明的具体实施方式中，所述的l优选为1、2、3、4、5或6。
所述PEG为相同或不同的-(CH 2CH 2O) m-，m的平均值为3-250的整数；优选的，所述m为68-250的整数；更优选的，所述m为68-227的整数。
所述C为CPPs，选自转录反式激活蛋白(Tat)、VP22、transportan、膜型两亲性肽(MAP)、信号转导肽和富含精氨酸序列肽；
优选的，所述C选自：LMWP、Tat48-60、Tat48-60-P10、CAI、HIV-TAT、MAP、 MPGα、M918、R6Pen、penetratin、Pep-1-K、ARF1-22、Tp10、POD、3-100个赖氨酸残基组成的聚赖氨酸、4-9个精氨酸残基组成的聚精氨酸；
更优选的，所述C为LMWP和由8个精氨酸组成的聚精氨酸。
所述D为药物分子，所述药物分子选自：小分子药物、染料、多肽、多肽核酸、蛋白质、抗体、质粒DNA、核酸、脂质体、噬菌体颗粒、超顺磁性粒子、荧光染剂、纳米粒、病毒、量子点、磁共振成像造影剂，特别是siRNA，尤其是Cytokine-siRNA；
优选的，所述D选自小分子药物、多肽、抗体、核酸，所述核酸包括核苷酸单体或寡聚核苷酸；核苷酸单体包括四种脱氧核糖核苷酸单体和四种荷塘核苷酸单体；寡聚核苷酸为取代寡核苷酸和非取代寡核苷酸，所述取代寡核苷酸为磷酰二胺吗啉代寡核苷酸，所述非取代寡核苷酸选自锁核酸、siRNA、microRNA、核酸适体、肽核酸、诱骗ODN、催化性RNA以及CpG二核苷酸中的任意一种；
更优选的，所述D选自单克隆抗体、寡聚核苷酸为长度为19-23bp的siRNA。
所述T为靶向基团，所述T选自：蛋白质、抗体、抗体片段或其衍生物、小分子肽、多肽、葡萄糖、半乳糖、叶酸、透明质酸；
优选的，所述抗体为单克隆抗体，所述抗体片段或其衍生物是Fv或Fab片段的单链。
在本发明的优选实施方式中，所述T选自：叶酸、RGD、cRGD、透明质酸、葡萄糖、半乳糖。
所述X为PEG连接CPPs的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、-三氮唑-、碳硫键或醚键中的一种或两种以上组成；
优选的，所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) j-S-(CH 2) j-中的一种或两种以上组合；
更优选的，所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) j-S-(CH 2) j-；
其中j为0-10的整数；优选的，j为0-5的整数；更优选的，j为0-3的整数；在本发明的具体实施方式中，j为0、1、2、3、4或5。
所述Y为PEG与药物分子D之间的连接键，所述连接键选自：二硫键、腙键、酰胺键、酯键、醚键、羰基键、硫酯键或巯基-马来酰亚胺；
优选的，所述Y选自：二硫键、腙键、酰胺键、酯键、硫酯键和巯基-马来酰亚胺，所述的二硫键和腙键用于细胞质释药；所述的酰胺键、酯键、硫酯键和巯基-马来酰亚胺用于细胞核内释药。
所述B为PEG与靶向基团的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、碳硫键或醚键中的一种或两种以上组成；
优选的，所述B选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) j-S-(CH 2) j-中的一种或两种以上组合；
更优选的，所述B选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) j-S-(CH 2) j-；
其中j为0-10的整数；优选的，j为0-5的整数；更优选的，j为0-3的整数；在本发明的具体实施方式中，j为0、1、2、3、4或5。
所述n为分支数或臂数，n≥3的整数；优选的，所述n为3-22的整数；更优选的，所述n为3-14的整数，最优选的，所述n为3-8整数。
所述k为连接CPPs端的分支数或臂数，1≤k≤n；优选的，所述k为1-14的整数；更优选的，所述k为1-6的整数；在本发明的实施方式中，所述k为1、2、4、6。
所述g为连接靶向基团的分支数或臂数，1≤g≤n；优选的，所述g为1-8的整数；更优选的，所述g为1-4的整数；在本发明的实施方式中，所述g为1、2、3、4。
在本发明的具体实施方式中，所述通式为Ⅳ的穿膜肽-多臂PEG-药物偶联物结构如下：
Figure PCTCN2019081057-appb-000009
Figure PCTCN2019081057-appb-000010
一种通式为Ⅴ的穿膜肽-多臂PEG-药物偶联物：
Figure PCTCN2019081057-appb-000011
所述R为中心分子，选自：多羟基结构、多氨基结构或多羧基结构；
优选的，所述R选自：季戊四醇或聚季戊四醇结构、丙三醇或聚丙三醇结构、甲基葡萄糖甙、蔗糖；在本发明的优选实施方式中，所述R选自：
Figure PCTCN2019081057-appb-000012
其中l为≥1且≤10的整数；优选的，l为≥1且≤10的整数，更优选，l为≥1且≤7的整数；在本发明的具体实施方式中，所述的l优选为1、2、3、4、5或6。
所述PEG为相同或不同的-(CH 2CH 2O) m-，m的平均值为3-250的整数；优选的，所述m为68-250的整数；更优选的，所述m为68-227的整数。
所述C为CPPs，选自转录反式激活蛋白(Tat)、VP22、transportan、膜型两亲性肽(MAP)、信号转导肽和富含精氨酸序列肽；
优选的，所述C选自：LMWP、Tat48-60、Tat48-60-P10、CAI、HIV-TAT、MAP、MPGα、M918、R6Pen、penetratin、Pep-1-K、ARF1-22、Tp10、POD、3-100个赖氨酸残基组成的聚赖氨酸、4-9个精氨酸残基组成的聚精氨酸；
更优选的，所述C为LMWP和由8个精氨酸组成的聚精氨酸。
所述D和D’分别独立的选自：小分子药物、染料、多肽、多肽核酸、蛋白质、抗体、质粒DNA、核酸、脂质体、噬菌体颗粒、超顺磁性粒子、荧光染剂、纳米粒、病毒、量子点、磁共振成像造影剂，特别是siRNA，尤其是Cytokine-siRNA；
优选的，所述D选自小分子药物；更优选的，所述D选自小分子药物，所述小分子药物分子量小于1000，如维生素C、乙酰水杨酸、乙酰氨基酚、对乙酰氨基酚等。
优选的，所述D’选自核酸，所述核酸包括核苷酸单体或寡聚核苷酸；核苷酸单体包括四种脱氧核糖核苷酸单体和四种荷塘核苷酸单体；寡聚核苷酸为取代寡核苷酸和非取代寡核苷酸，所述取代寡核苷酸为磷酰二胺吗啉代寡核苷酸，所述非取代寡核苷酸选自锁核酸、siRNA、microRNA、核酸适体、肽核酸、诱骗ODN、催化性RNA以及CpG二核苷酸中的任意一种；
更优选的，所述D’选自单克隆抗体、寡聚核苷酸为长度为19-23bp的siRNA。
所述X为PEG连接CPPs的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、-三氮唑-、碳硫键或醚键中的一种或两种以上组成；
优选的，所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) j-S-(CH 2) j-中的一种或两种以上组合；
更优选的，所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) j-S-(CH 2) j-；
其中j为0-10的整数；优选的，j为0-5的整数；更优选的，j为0-3的整数；在本发明的具体实施方式中，j为0、1、2、3、4或5。
所述Y为PEG与药物分子D之间的连接键，所述连接键选自：二硫键、腙键、酰胺键、酯键、醚键、羰基键、硫酯键或巯基-马来酰亚胺；
优选的，所述Y选自：二硫键、腙键、酰胺键、酯键、硫酯键和巯基-马来酰亚胺，所述的二硫键和腙键用于细胞质释药；所述的酰胺键、酯键、硫酯键和巯基-马来酰亚胺用于细胞核内释药。
所述Z为PEG与药物分子D’之间的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、碳硫键或醚键中的一种或两种以上组成；
优选的，所述Z选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) j-S-(CH 2) j-中的一种或两种以上组合；
更优选的，所述Z选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) j-S-(CH 2) j-；
其中j为0-10的整数；优选的，j为0-5的整数；更优选的，j为0-3的整数；在本发明的具体实施方式中，j为0、1、2、3、4或5。
所述n为分支数或臂数，n≥3的整数；优选的，所述n为3-22的整数；更优选的，所述n为3-14的整数，最优选的，所述n为3-8整数。
所述k为连接CPPs端的分支数或臂数，1≤k≤n；优选的，所述k为1-14的整数；更优选的，所述k为1-6的整数；在本发明的实施方式中，所述k为1、2、4、6。
所述p为连接药物分子D’的分支数或臂数，1≤p≤n；优选的，所述p为1-8的整数；更优选的，所述p为1-4的整数；在本发明的实施方式中，所述p为1、2、3、4。
在本发明的具体实施方式中，所述通式为Ⅴ的穿膜肽-多臂PEG-药物偶联物结构如下：
Figure PCTCN2019081057-appb-000013
在本发明中，所述PEG分子量为1000-80000Da；优选的，所述PEG分子量为3000-20000Da；更优选的，所述的PEG分子量为3000-10000Da；在本发明的最优选的实施方案中，所述的PEG分子量可以为3000Da、5000Da、10000Da、20000Da。
本发明还提供一种药物组合物，所述的药物组合物包括通式为Ⅰ、Ⅱ、Ⅲ、Ⅳ或Ⅴ的穿膜肽-多臂PEG-药物偶联物。
优选的，所述药物组合物中还包括一种或多种药剂学上可接受的辅料，所述辅料选自：载体、稀释剂、粘合剂、润滑剂、润湿剂。
优选的，所述药物组合物的剂型包括：片剂、胶囊、丸剂、注射剂、乳剂、微乳剂、纳米颗粒、吸入剂、含片、凝胶剂、粉剂、栓剂、悬乳液、乳膏剂、胶冻剂、喷雾剂。
优选的，所述药物组合物可采取的给药方式包括：口服、皮下注射、肌肉注射、静脉注射、直肠给药、阴道给药、鼻腔给药、透皮给药、结膜下给药、眼球内给药、眼眶给药、眼球后给药、视网膜给药、脉络膜给药、鞘内注射。
本发明所述通式为Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ的穿膜肽-多臂PEG-药物偶联物在制备靶向药物中的应用，所述的药物用于疾病的诊断和治疗；优选的，所述的疾病选自肿瘤、肺炎、哮喘、肺纤维化、病毒感染、肝炎、眼部黄斑性病变等；优选的，所述疾病选自眼部黄斑性病变、哮喘、肺纤维化。
本发明所提供的通式为Ⅰ、Ⅱ、Ⅲ/Ⅳ和Ⅴ的穿膜肽-多臂PEG-药物偶联物，与直链型PEG-穿膜肽偶联物相比，多臂PEG具有多个端基，有多个功能基团的引入点，可以连接多个不同的活性基团，避免了直链型PEG连接位点有限，应用范围小、药物负载量低的问题。另外，本发明所提供的穿膜肽-多臂PEG-药物偶联物具有靶向性，可根据具体治疗需要，在穿膜肽上、药物分子上、或PEG链端连接靶向基团，使药物靶向地进入致病细胞，达到精准治疗目的。
本发明所述的CPPs的氨基酸序列如下：
LMWP            VSRRRRRRGGRRRR
Tat48-60        GRKKRRQRRRPPQ
Tat48-60-P10    GRKKRRQRRRPPQRQTSMTDFYHSKRRLIFS
CAI             ITFEDLLDYYGP-NH 2
HIV-TAT         RKKRRQRRR
MAP             KLALKLALKALKAALKLA-NH 2
MPGα            Ac-GALFLAFLAAALSCMGLWSQPKKKRKV-Cya
M918            MVTVLFRRLRIRRACGPPRVRV-NH 2
R6Pen           RRRRRRRQIKIWFQNRRMKWKK
Penetratin      RQIKIWFQNRRMKWKK
Pep-1-K         KKTWWKTWWTKWSQPKKKRKV
ARF1-22         MVRRFLVTLRIRRACGPPRVRV-NH 2
Tp10            AGYLLGKINLKALAALAKKIL-NH 2
POD             GGG(ARKKAAKA) 4
具体实施方式
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 四臂PEG-LMWP偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000014
将LMWP溶解于20mM KH2PO4缓冲溶液中，将4arm PEG-1arm NHS-3arm Opss溶解于DMSO中，然后滴加到缓冲溶液中，室温下反应2小时，过滤，用肝素柱纯化得产品产品通过冷冻干燥或沉淀方式得到；
实施例2 四臂PEG-LMWP-MAL偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000015
将上述得到的4arm PEG-1arm LMWP-3arm OPSS溶解在二氯甲烷中，加入适量的TEA和MAL-NH2，室温下反应12小时，浓缩反应液，纯化、冷冻干燥或用异丙醇将产品沉淀出来；
实施例3 四臂PEG-LMWP-MAL-RGD偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000016
将上述得到的4arm PEG-1arm LMWP-MAL-3arm OPSS溶解在二氯甲烷中，加入适量的RGD，室温下反应12小时，浓缩反应液，纯化、冷冻干燥或用用异丙醇将产品沉淀出来；
实施例4 四臂PEG-LMWP-MAL-RGD-3arm-VEGF-siRNA偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000017
向上述得到的4arm PEG-1arm LMWP-MAL-RGD-3arm OPSS中加入1M的DTT溶液，室温下反应，得到4arm PEG-1arm LMWP-MAL-RGD-3arm Thiol，然后将突变的SH-VEGF-siRNA溶于10mM KH2PO4，0.15M NaCl中，将其滴加至适量的用肝素柱纯化过的4arm PEG-1arm LMWP-MAL-RGD-3arm Thiol中，边滴加边搅拌，将上述混合物连续搅拌反应2小时，反应完毕后，未反应的SH-VEGF-siRNA用阳离子柱纯化除去。用冷冻干燥或沉淀的方式得到产品，产品储于-20℃保存。
其中SH-VEGF-siRNA具有的正义链和反义链序列如下：
正义链：5＇-GAUAGAGCAAGACAAGAAAUU-3＇
反义链：3＇-UUCUAUCUCGUUCUGUUCUUU-5＇
实施例5 四臂PEG-LMWP-(VEGF-siRNA)3偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000018
将上述得到的4arm PEG-1arm LMWP-3arm OPSS溶液，直接滴加入含有一定量的SH-VEGF-siRNA 20mM NaH2PO4 1mM EDTA PH＝6.9的缓冲溶液中，边滴边搅拌，体系在40度条件下反应30min至1h，凝胶电泳检测SH-VEGF-siRNA反应完毕，停止反应。用DEAE柱对反应液进行纯化，流动相体系为A相：20mM NaH2PO4 1mM EDTA PH＝6.9的缓冲溶液；B相：20mM NaH2PO4 2MNaCl 1mM EDTA PH＝6.9的缓冲溶液；在45％B相时收集样品，凝胶电泳显示产品为一个条带。将样品用截留分子量为3000的超滤离心管进行脱盐，用冷冻干燥或沉淀的方式得到产品，产品储于-20℃保存。
实施例6 四臂PEG-(LMWP)2偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000019
将LMWP溶解于20mM KH2PO4缓冲溶液中，将4arm PEG-2arm NHS-2arm Opss溶解于DMSO中，然后滴加到缓冲溶液中，室温下反应2小时，过滤，用肝素柱纯化得产品 产品通过冷冻干燥方式得到，也可直接用溶液进行下一步反应；
实施例7 四臂PEG-(LMWP)2-(VEGF-siRNA)2偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000020
将上述得到的4arm PEG-2arm LMWP-2arm OPSS溶液，直接滴加入含有一定量的SH-VEGF-siRNA 20mM NaH2PO4 1mM EDTA PH＝6.9的缓冲溶液中，边滴边搅拌，体系在40度条件下反应30min至1h，凝胶电泳检测SH-VEGF-siRNA反应完毕，停止反应。用DEAE柱对反应液进行纯化，流动相体系为A相：20mM NaH2PO4 1mM EDTA PH＝6.9的缓冲溶液；B相：20mM NaH2PO4 2MNaCl 1mM EDTA PH＝6.9的缓冲溶液；在45％B相时收集样品，凝胶电泳显示产品为一个条带。将样品用截留分子量为3000的超滤离心管进行脱盐，用冷冻干燥或沉淀的方式得到产品，产品储于-20℃保存。
实施例8 四臂PEG-(LMWP)3偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000021
将LMWP溶解于20mM KH2PO4缓冲溶液中，将4arm PEG-3arm NHS-1arm Opss溶解于DMSO中，然后滴加到缓冲溶液中，室温下反应2小时，过滤，用肝素柱纯化得产品产品通过冷冻干燥方式得到，也可直接用溶液进行下一步反应；
实施例9 四臂PEG-(LMWP)3-VEGF-siRNA偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000022
将上述得到的4arm PEG-3arm LMWP-1arm OPSS溶液，直接滴加入含有一定量的SH-VEGF-siRNA 20mM NaH2PO4 1mM EDTA PH＝6.9的缓冲溶液中，边滴边搅拌，体系在40度条件下反应30min至1h，凝胶电泳检测SH-VEGF-siRNA反应完毕，停止反应。用DEAE柱对反应液进行纯化，流动相体系为A相：20mM NaH2PO4 1mM EDTA PH＝6.9的缓冲溶液；B相：20mM NaH2PO4 2MNaCl 1mM EDTA PH＝6.9的缓冲溶液；在45％B相时收集样品，凝胶电泳显示产品为一个条带。将样品用截留分子量为3000的超滤离心管进行脱盐，用冷冻干燥或沉淀的方式得到产品，产品储于-20℃保存。
实施例10 八臂PEG-LMWP偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000023
将LMWP溶解于20mM KH2PO4缓冲溶液中，将8arm PEG-1arm NHS-7arm Opss溶解于DMSO中，然后滴加到缓冲溶液中，室温下反应2小时，过滤，用肝素柱纯化得产品产品通过冷冻干燥或沉淀方式得到；
实施例11 八臂PEG-(LMWP)1-(VEGF-siRNA)7偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000024
将上述得到的8arm PEG-1arm LMWP-7arm OPSS溶液，直接滴加入含有一定量的SH-VEGF-siRNA 20mM NaH2PO4 1mM EDTA PH＝6.9的缓冲溶液中，边滴边搅拌，体系在40度条件下反应30min至1h，凝胶电泳检测SH-VEGF-siRNA反应完毕，停止反应。用DEAE柱对反应液进行纯化，流动相体系为A相：20mM NaH2PO4 1mM EDTA PH＝6.9的缓冲溶液；B相：20mM NaH2PO4 2MNaCl 1mM EDTA PH＝6.9的缓冲溶液；在45％B相时收集样品，凝胶电泳显示产品为一个条带。将样品用截留分子量为3000的超滤离心管进行脱盐，用冷冻干燥或沉淀的方式得到产品，产品储于-20℃保存。
实施例12 八臂PEG-(LMWP)2偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000025
将LMWP溶解于20mM KH2PO4缓冲溶液中，将8arm PEG-2arm NHS-6arm Opss溶解于DMSO中，然后滴加到缓冲溶液中，室温下反应2小时，过滤，用肝素柱纯化得产品产品通过冷冻干燥或沉淀方式得到；
实施例13 八臂PEG-(LMWP)2-(VEGF-siRNA)6偶联物的制备方法(MW10000)
Figure PCTCN2019081057-appb-000026
将上述得到的8arm PEG-2arm LMWP-6arm OPSS溶液，直接滴加入含有一定量的SH-VEGF-siRNA 20mM NaH2PO4 1mM EDTA PH＝6.9的缓冲溶液中，边滴边搅拌，体系在40度条件下反应30min至1h，凝胶电泳检测SH-VEGF-siRNA反应完毕，停止反应。用DEAE柱对反应液进行纯化，流动相体系为A相：20mM NaH2PO4 1mM EDTA PH＝6.9的缓冲溶液；B相：20mM NaH2PO4 2MNaCl 1mM EDTA PH＝6.9的缓冲溶液；在45％B相时收集样品，凝胶电泳显示产品为一个条带。将样品用截留分子量为3000的超滤离心管进行脱盐，用冷冻干燥或沉淀的方式得到产品，产品储于-20℃保存。
实施例14 LMWP-PEG5000-VEGF-siRNA与四臂PEG-LMWP-(VEGF-siRNA)3(MW 10000)细胞效果抑制比较
LMWP-PEG5000-VEGF-siRNA与实施例5制备得到的四臂PEG-LMWP-(VEGF-siRNA) 3(MW 10000)在293T细胞上进行细胞转染，用RT-PCR检测样品中VEGFA的表达情况，结果是四臂PEG-LMWP-(VEGF-siRNA) 3(MW 10000)的细胞效果明显好于LMWP-PEG5000-VEGF-siRNA。
以上具体实施方式只是对本发明内容的示意性说明，不代表本发明内容的限制。本领域技术人员可以想到的是本发明中具体结构可以有其它的变化形式。

Claims (19)

  1. 一种通式为Ⅰ的穿膜肽-多臂PEG-药物偶联物：
    Figure PCTCN2019081057-appb-100001
    所述R为中心分子，选自：多羟基结构、多氨基结构或多羧基结构；
    所述PEG为相同或不同的-(CH 2CH 2O) m-，m的平均值为3-250的整数；
    所述C为CPPs，选自转录反式激活蛋白、VP22、transportan、膜型两亲性肽、信号转导肽和富含精氨酸序列肽；
    所述D为药物分子，所述药物分子选自：小分子药物、染料、多肽、抗体、质粒DNA、核酸、脂质体、噬菌体颗粒、超顺磁性粒子、荧光染剂、纳米粒、病毒、量子点、磁共振成像造影剂；
    所述X为PEG连接CPPs的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、-三氮唑-、碳硫键或醚键中的一种或两种以上组成；
    所述Y为PEG与药物分子D之间的连接键，所述连接键选自：二硫键、腙键、酰胺键、酯键、醚键、羰基键、硫酯键或巯基-马来酰亚胺；
    所述n为分支数或臂数，n≥3的整数；所述k为连接CPPs端的分支数或臂数，1≤k≤n。
  2. 根据权利要求1所述的穿膜肽-多臂PEG-药物偶联物，其特征在于，所述R选自：季戊四醇或聚季戊四醇结构、丙三醇或聚丙三醇结构、甲基葡萄糖甙、蔗糖；
    所述C选自：LMWP、Tat48-60、Tat48-60-P10、CAI、HIV-TAT、MAP、MPGα、M918、R6Pen、penetratin、Pep-1-K、ARF1-22、Tp10、POD、3-100个赖氨酸残基组成的聚赖氨酸、4-9个精氨酸残基组成的聚精氨酸；
    所述D选自小分子药物、多肽、抗体、核酸；
    所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-，-三氮唑-、巯基-马来酰亚胺键中的一种或两种以上组合；其中j为0-10的整数；
    所述Y选自：二硫键、腙键、酰胺键、酯键、硫酯键和巯基-马来酰亚胺；
    所述n为3-22的整数；所述k为1-14的整数。
  3. 根据权利要求2所述的穿膜肽-多臂PEG-药物偶联物，其特征在于，所述R选自：
    Figure PCTCN2019081057-appb-100002
    其中l为≥1且≤10的整数；
    所述C为LMWP或由8个精氨酸组成的聚精氨酸；
    所述D选自奥马珠单抗、尼达尼布、贝伐珠单抗、派姆单抗、曲妥珠单抗、纳武单抗，VEGF-siRNA、IL-v-siRNA、Syk-siRNA、GATA-3-siRNA，其中v选自4，5，8，13；
    所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-，-三氮唑-、巯基-马来酰亚胺键，其中j为0-5的整数；
    所述n为3-14的整数；所述k为1-6的整数。
  4. 权利要求1所述的通式为Ⅰ的穿膜肽-多臂PEG-药物偶联物结构为：
    Figure PCTCN2019081057-appb-100003
  5. 一种通式为Ⅱ或Ⅲ的穿膜肽-多臂PEG-药物偶联物：
    Figure PCTCN2019081057-appb-100004
    所述R为中心分子，选自：多羟基结构、多氨基结构或多羧基结构；
    所述PEG为相同或不同的-(CH 2CH 2O) m-，m的平均值为3-250的整数；
    所述C为CPPs，选自转录反式激活蛋白、VP22、transportan、膜型两亲性肽、信号转导肽、富含精氨酸序列肽；
    所述D为药物分子，所述药物分子选自：小分子药物、染料、多肽、抗体、质粒DNA、核酸、脂质体、噬菌体颗粒、超顺磁性粒子、荧光染剂、纳米粒、病毒、量子点、磁共振成像造影剂；
    所述T为靶向基团，所述T选自：蛋白质、抗体、抗体片段或其衍生物、小分子肽、多肽、葡萄糖、半乳糖、叶酸、透明质酸；
    所述X为PEG连接CPPs的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、-三氮唑-、碳硫键或醚键中的一种或两种以上组成；
    所述Y为PEG与药物分子D之间的连接键，所述连接键选自：二硫键、腙键、酰胺键、酯键、醚键、羰基键、硫酯键或巯基-马来酰亚胺；
    所述B为穿膜肽或药物分子与靶向基团的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、碳硫键或醚键中的一种或两种以上组成；
    所述n为分支数或臂数，n≥3的整数；所述k为连接CPPs端的分支数或臂数，1≤k≤n。
  6. 根据权利要求5所述的穿膜肽-多臂PEG-药物偶联物，其特征在于，所述R选自：季戊四醇或聚季戊四醇结构、丙三醇或聚丙三醇结构、甲基葡萄糖甙、蔗糖；
    所述C选自：LMWP、Tat48-60、Tat48-60-P10、CAI、HIV-TAT、MAP、MPGα、M918、R6Pen、penetratin、Pep-1-K、ARF1-22、Tp10、POD、3-100个赖氨酸残基组成的聚赖氨酸、4-9个精氨酸残基组成的聚精氨酸；
    所述D选自小分子药物、多肽、抗体、核酸；
    所述T中的抗体为单克隆抗体，所述抗体片段或其衍生物是Fv或Fab片段的单链；
    所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-中的一种或两种以上组合；其中j为0-10的整数；
    所述Y选自：二硫键、腙键、酰胺键、酯键、硫酯键和巯基-马来酰亚胺；
    所述B选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-中的一种或两种以上组合，其中j为0-10的整数；
    所述n为3-22的整数；所述k为1-14的整数。
  7. 根据权利要求6所述的穿膜肽-多臂PEG-药物偶联物，其特征在于，所述R选自：
    Figure PCTCN2019081057-appb-100005
    其中l为≥1且≤10的整数；
    所述C为LMWP和由8个精氨酸组成的聚精氨酸；
    所述D选自寡聚核苷酸长度为19-23bp的siRNA，；
    所述T选自：叶酸、RGD、cRGD、透明质酸、葡萄糖、半乳糖；
    所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-，其中j为0-5的整数；
    所述B选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-，其中j为0-5的整数；
    所述n为3-14的整数；所述k为1-6的整数。
  8. 权利要求5所述的通式为Ⅱ或Ⅲ的穿膜肽-多臂PEG-药物偶联物结构为：
    Figure PCTCN2019081057-appb-100006
    Figure PCTCN2019081057-appb-100007
  9. 一种通式为Ⅳ的穿膜肽-多臂PEG-药物偶联物：
    Figure PCTCN2019081057-appb-100008
    所述R为中心分子，选自：多羟基结构、多氨基结构或多羧基结构；
    所述PEG为相同或不同的-(CH 2CH 2O) m-，m的平均值为3-250的整数；
    所述C为CPPs，选自转录反式激活蛋白、VP22、transportan、膜型两亲性肽、信号转导肽、富含精氨酸序列肽；
    所述D为药物分子，所述药物分子选自：小分子药物、染料、多肽、抗体、质粒DNA、核酸、脂质体、噬菌体颗粒、超顺磁性粒子、荧光染剂、纳米粒、病毒、量子点、磁共振成像造影剂；
    所述T为靶向基团，所述T选自：蛋白质、抗体、抗体片段或其衍生物、小分子肽、多肽、葡萄糖、半乳糖、叶酸、透明质酸；
    所述X为PEG连接CPPs的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、-三氮唑-、碳硫键或醚键中的一种或两种以上组成；
    所述Y为PEG与药物分子D之间的连接键，所述连接键选自：二硫键、腙键、酰胺键、酯键、醚键、羰基键、硫酯键或巯基-马来酰亚胺；
    所述B为PEG与靶向基团的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、碳硫键或醚键中的一种或两种以上组成；
    所述n为分支数或臂数，n≥3的整数；所述k为连接CPPs端的分支数或臂数，1≤k≤n；所述g为连接靶向基团的分支数或臂数，1≤g≤n。
  10. 根据权利要求9所述的穿膜肽-多臂PEG-药物偶联物，其特征在于，所述R选自：季戊四醇或聚季戊四醇结构、丙三醇或聚丙三醇结构、甲基葡萄糖甙、蔗糖；
    所述C选自：LMWP、Tat48-60、Tat48-60-P10、CAI、HIV-TAT、MAP、MPGα、M918、R6Pen、penetratin、Pep-1-K、ARF1-22、Tp10、POD、3-100个赖氨酸残基组成的聚赖氨酸、4-9个精氨酸残基组成的聚精氨酸；
    所述D选自小分子药物、多肽、抗体、核酸；
    所述T中的抗体为单克隆抗体，所述抗体片段或其衍生物是Fv或Fab片段的单链；
    所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-中的一种或两种以上组合；其中j为0-10的整数；
    所述Y选自：二硫键、腙键、酰胺键、酯键、硫酯键和巯基-马来酰亚胺；
    所述B选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-中的一种或两种以上组合，其中j为0-10的整数；
    所述n为3-22的整数；所述k为1-14的整数；所述g为1-8的整数。
  11. 根据权利要求10所述的穿膜肽-多臂PEG-药物偶联物，其特征在于，所述R选自：
    Figure PCTCN2019081057-appb-100009
    其中l为≥1且≤10的整数；
    所述C为LMWP和由8个精氨酸组成的聚精氨酸；
    所述D选自单克隆抗体、寡聚核苷酸为长度为19-23bp的siRNA；
    所述T选自：叶酸、RGD、cRGD、透明质酸、葡萄糖、半乳糖；
    所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-，其中j为0-5的整数；
    所述B选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-，其中j为0-5的整数；
    所述n为3-14的整数；所述k为1-6的整数；所述g为1-4的整数。
  12. 权利要求9所述的通式为Ⅳ的穿膜肽-多臂PEG-药物偶联物，结构为：
    Figure PCTCN2019081057-appb-100010
  13. 一种通式为Ⅴ的穿膜肽-多臂PEG-药物偶联物：
    Figure PCTCN2019081057-appb-100011
    所述R为中心分子，选自：多羟基结构、多氨基结构或多羧基结构；
    所述PEG为相同或不同的-(CH 2CH 2O) m-，m的平均值为3-250的整数；
    所述C为CPPs，选自转录反式激活蛋白(Tat)、VP22、transportan、膜型两亲性肽(MAP)、信号转导肽、富含精氨酸序列肽；
    所述D和D’分别独立的选自：小分子药物、染料、多肽、抗体、质粒DNA、核酸、脂质体、噬菌体颗粒、超顺磁性粒子、荧光染剂、纳米粒、病毒、量子点、磁共振成像造影剂；
    所述X为PEG连接CPPs的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、-三氮唑-、碳硫键或醚键中的一种或两种以上组成；
    所述Y为PEG与药物分子D之间的连接键，所述连接键选自：二硫键、腙键、酰胺键、酯键、醚键、羰基键、硫酯键或巯基-马来酰亚胺；
    所述Z为PEG与药物分子D’之间的连接键，所述连接键由酰胺键、二硫键、腙键、酯键、硫酯键、巯基-马来酰亚胺键、碳硫键或醚键中的一种或两种以上组成；
    所述n为分支数或臂数，n≥3的整数；所述k为连接CPPs端的分支数或臂数，1≤k≤n；所述p为连接药物分子D’的分支数或臂数，1≤p≤n。
  14. 根据权利要求13所述的穿膜肽-多臂PEG-药物偶联物，其特征在于，所述R选自：季戊四醇或聚季戊四醇结构、丙三醇或聚丙三醇结构、甲基葡萄糖甙、蔗糖；
    所述C选自：LMWP、Tat48-60、Tat48-60-P10、CAI、HIV-TAT、MAP、MPGα、M918、R6Pen、penetratin、Pep-1-K、ARF1-22、Tp10、POD、3-100个赖氨酸残基组成的聚赖氨酸、4-9个精氨酸残基组成的聚精氨酸；
    所述D选自小分子药物；
    所述D’选自核酸；
    所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-中的一种或两种以上组合；其中j为0-10的整数；
    所述Y选自：二硫键、腙键、酰胺键、酯键、硫酯键和巯基-马来酰亚胺；
    所述Z选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、 -(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-中的一种或两种以上组合，其中j为0-10的整数；
    所述n为3-22的整数；所述k为1-14的整数；所述p为1-8的整数。
  15. 根据权利要求13所述的穿膜肽-多臂PEG-药物偶联物，其特征在于，所述R选自：
    Figure PCTCN2019081057-appb-100012
    其中l为≥1且≤10的整数；
    所述C为LMWP和由8个精氨酸组成的聚精氨酸；
    所述D选自小分子药物；
    所述D’选自单克隆抗体、寡聚核苷酸为长度为19-23bp的siRNA；
    所述X选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-，其中j为0-5的整数；
    所述Z选自-(CH 2) jCONH(CH 2) j-、-(CH 2) j-S-S-(CH 2) j-、-(CH 2) jNH-N＝C(CH 2) j-、-(CH 2) jCOO(CH 2) j-、-(CH 2) j-S-(CH 2) j-，其中j为0-5的整数；
    所述n为3-14的整数；所述k为1-6的整数；所述p为1-4的整数。
  16. 权利要求13所述的通式为Ⅴ的穿膜肽-多臂PEG-药物偶联物，结构为：
    Figure PCTCN2019081057-appb-100013
  17. 一种药物组合物，所述药物组合物包括权利要求1-16任一所述的穿膜肽-多臂PEG- 药物偶联物。
  18. 权利要求1-16任一所述的穿膜肽-多臂PEG-药物偶联物在制备靶向药物中的应用，所述药物用于疾病的诊断和治疗；所述的疾病选自肿瘤、肺炎、哮喘、肺纤维化、病毒感染、肝炎、眼部黄斑性病变。
  19. 根据权利要求18所述的穿膜肽-多臂PEG-药物偶联物在制备靶向药物中的应用，其特征在于，所述疾病选自眼部黄斑性病变、哮喘、肺纤维化。
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