WO2023016410A1 - Anti-angiogenic drug - Google Patents

Anti-angiogenic drug Download PDF

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WO2023016410A1
WO2023016410A1 PCT/CN2022/110868 CN2022110868W WO2023016410A1 WO 2023016410 A1 WO2023016410 A1 WO 2023016410A1 CN 2022110868 W CN2022110868 W CN 2022110868W WO 2023016410 A1 WO2023016410 A1 WO 2023016410A1
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endostatin
human endostatin
reduced human
modified
reduced
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PCT/CN2022/110868
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French (fr)
Chinese (zh)
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刘鹏
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刘鹏
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • 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
    • 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
    • A61K47/56—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
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a reduced human endostatin or an analog thereof and a modified reduced human endostatin or an analog thereof.
  • the present invention also relates to a modified protein or polypeptide, which can be modified to make the water-insoluble protein or polypeptide water-soluble under the condition of physiological pH value, thereby improving its druggability.
  • Endostatin is an enzyme-cleaved product with a molecular weight of 20 kDa at the carboxy-terminal of collagen XVIII.
  • Professor Judah Folkman of Harvard University and others discovered this protein in the culture of hemangioendothelioma cells, which has the activity of inhibiting the proliferation, migration and angiogenesis of vascular endothelial cells (O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J: Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88:277–85, 1997).
  • the recombinant human endostatin prepared by genetic engineering can be used as a tumor treatment drug.
  • the trade name of the only recombinant human endostatin drug approved for marketing at present is "Endostar”.
  • Endostar The trade name of the only recombinant human endostatin drug approved for marketing at present.
  • the initial research found that endostatin has extremely high anti-angiogenic activity and anti-tumor activity, but the subsequent basic research and clinical research showed that the activity was not ideal (B.Kim Lee Sim, Nicholas J.MacDonald and Edward R. Gubish: Angiostatin and Endostatin: Endogenous Inhibitors of Tumor Growth. Cancer and Metastasis Reviews 19:181–190, 2000). Therefore, in practical applications, endostatins with better antitumor activity are needed.
  • the purpose of the present invention is to provide a reduced human endostatin or its analog and modified reduced human endostatin or its analog, the reduced human endostatin or its analog Compared with the existing oxidized human endostatin, the modified reduced human endostatin or its analogues have better anti-angiogenesis and anti-tumor activities.
  • the first aspect of the present invention provides a reduced state of human endostatin or its analogs, the reduced state of human endostatin contains at most a pair of disulfide bonds, the disulfide bond consists of Sulfhydryl formation on two cysteine residues of the human endostatin molecule.
  • the reduced human endostatin analogs include at least one or a combination of the following:
  • the reduced human endostatin analog contains at most one pair of disulfide bonds.
  • amino acid sequence of the reduced human endostatin is shown in SEQ ID NO: 1, 2, 3 or 4.
  • amino acid sequence of the reduced human endostatin is as shown in SEQ ID NO: 1, wherein the formation of disulfide bonds includes:
  • Cys135 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys165 residue;
  • the second aspect of the present invention also provides a modified reduced human endostatin or its analogs, the reduced human endostatin contains at most one pair of disulfide bonds, and the disulfide bonds are formed by human vascular endothelial Sulfhydryl groups formed on two cysteine residues of the inhibin molecule.
  • the reduced human endostatin analogs include at least one or a combination of the following:
  • the reduced human endostatin analog contains at most one pair of disulfide bonds.
  • amino acid sequence of the reduced human endostatin is shown in SEQ ID NO: 1, 2, 3 or 4.
  • amino acid sequence of the reduced human endostatin is shown in SEQ ID NO: 2 or 4.
  • amino acid sequence of the reduced human endostatin is as shown in SEQ ID NO: 1, wherein the formation of disulfide bonds includes:
  • Cys135 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys165 residue;
  • the modification is selected from any one or combination of high molecular polymers, protein molecules or fragments thereof, small molecular substances.
  • protein molecules or fragments thereof include albumin, immunoglobulins, cytokines or fragments thereof, preferably Fc fragments of immunoglobulins.
  • the reduced human endostatin or its analogs modified by the immunoglobulin Fc fragment can be obtained through chemical modification or fusion expression.
  • the reduced human endostatin modified by the immunoglobulin Fc fragment is expressed by fusion, the reduced human endostatin modified by the immunoglobulin Fc fragment is the formation of the immunoglobulin Fc fragment and the reduced human endostatin fusion protein.
  • the high molecular polymer is polyethylene glycol.
  • amino acid sequence of the reduced human endostatin is shown in SEQ ID NO: 2 or 4.
  • one reduced human endostatin or its analogue molecule is coupled to one or more polyethylene glycol molecules.
  • the site where the reduced human endostatin or its analogues are coupled to the polyethylene glycol is the site of the reduced human endostatin or its analogues.
  • the site where the reduced human endostatin or its analogs are coupled to the polyethylene glycol is the reduced human endostatin or its analogs N terminal ⁇ -amino group.
  • said polyethylene glycol molecules have an average molecular weight between 1,000 and 100,000 Daltons.
  • said polyethylene glycol molecules have an average molecular weight between 5,000 and 40,000 Daltons.
  • the third aspect of the present invention provides a pharmaceutical composition, which comprises the above-mentioned reduced human endostatin or its analogue or the above-mentioned modified reduced human endostatin or its analogue, and pharmaceutically acceptable Carrier.
  • the pharmaceutical composition is a sustained-release preparation.
  • the fourth aspect of the present invention provides a modified protein or polypeptide, the protein or polypeptide is insoluble in water under the condition of physiological pH value, and after modification, it is soluble in water under the condition of physiological pH value; the modification is selected from Any one or a combination of high molecular weight polymers, protein molecules or their fragments, small molecular substances.
  • the fifth aspect of the present invention provides the use of animal-derived vascular endostatin in the preparation of veterinary medicine.
  • Fig. 1 is the electrophoresis figure of the reduced recombinant human endostatin of reducing state detected by SDS-PAGE electrophoresis and the reduced recombinant human endostatin of polyethylene glycol (PEG); Wherein, L1 and L2 are polyethylene glycol (PEG) ) modified reduced recombinant human endostatin, L3 and L4 are reduced recombinant human endostatin.
  • PEG polyethylene glycol
  • Fig. 2 is the comparison chart of the activity that the reduction state recombinant human endostatin of PEG modification, endostar (Endostar) and the endostar (Endostar) of PEG modification inhibit HMEC cell (human microvascular endothelial cell) migration
  • B is blank
  • L1 is that the endostar that administration concentration is 10 ⁇ g/ml inhibits the activity of HMEC cell migration
  • L2 is that the endostar (PEG-Endostar) that administration concentration is 10 ⁇ g/ml is modified inhibits
  • L3 is the activity that the reduced state recombinant human endostatin (nES) modified by polyethylene glycol with the administration concentration of 1 ⁇ g/ml inhibits the activity of HMEC cell migration
  • L4 is the activity of the administration concentration of 2 ⁇ g/ml
  • the reduced state recombinant human endostatin (nES) modified by polyethylene glycol inhibits the activity of
  • Fig. 3 is each component, endostar and endostatin obtained by purifying polyethylene glycol-modified reduced recombinant human endostatin with cation exchange chromatography (the medium is sulfopropyl sephadex (abbreviated as SP)).
  • a comparison chart of the activity of polyethylene glycol-modified Endostar in inhibiting HMEC cell migration where, B is blank, L1 is the activity of Endostar at a concentration of 10 ⁇ g/ml in inhibiting HMEC cell migration, and L2 is the activity of Endostar at a concentration of 10 ⁇ g Endostar modified by polyethylene glycol/ml inhibits the activity of HMEC cell migration, L3 is the activity of nES (FT (effluent)) inhibiting HMEC cell migration, L4 is nES (25mS/cm ) component inhibits the activity of HMEC cell migration, L5 is the activity of the nES (30mS/cm) component whose administration concentration is 2 ⁇ g/ml inhibits HMEC cell migration, and L6 is the nES (40mS/cm ) component inhibits the activity of HMEC cell migration.
  • B blank
  • L1 is the activity of Endostar at a concentration of 10 ⁇ g/ml in inhibiting
  • Fig. 4 is the comparison figure of the nES (25mS/cm) component of different administration concentrations, endostar and polyethylene glycol-modified endostar inhibiting the activity of HMEC cell migration;
  • B is blank, and L1 is that administration concentration is Endostar 10 ⁇ g/ml inhibits HMEC cell migration, L2 is the activity of polyethylene glycol-modified Endostar at a concentration of 10 ⁇ g/ml to inhibit HMEC cell migration, L3 is nES at a concentration of 0.1ug/ml (25mS/cm) component inhibits the activity of HMEC cell migration, L4 is the activity of nES (25mS/cm) component with an administration concentration of 0.2 ⁇ g/ml to inhibit HMEC cell migration, L5 is the administration concentration of 0.5 ⁇ g/ml The nES (25mS/cm) component of L6 inhibits the activity of HMEC cell migration, and the nES (25mS/cm) component with
  • Figure 5 is a comparison chart of the activity of blank control or polyethylene glycol-modified reduced recombinant human vascular endostatin inhibiting the proliferation of HMEC cells; wherein, Figure a is the activity of the blank control to inhibit the proliferation of HMEC cells, and Figure b is the activity of polyethylene glycol-modified recombinant human vascular endostatin Alcohol-modified reduced recombinant human endostatin inhibits the proliferation of HMEC cells.
  • Figure 6 is a schematic diagram of the spatial structure of the existing oxidized human vascular endostatin
  • Fig. 7 is a schematic diagram of disulfide bond pairing of existing oxidized human endostatin.
  • neovascularization refers to the generation of new capillaries on top of existing blood vessels.
  • Numerous diseases are known to be associated with neovascularization, such as tumors and macular degeneration. Taking tumors as an example, the growth and migration of tumors depend on the formation of new blood vessels.
  • Targeting microvascular endothelial cells in tumors as a target for cancer therapy offers a therapeutic modality for treating tumors.
  • HMEC Human Microvascular Endothelial Cell line
  • HUVEC Human Umbilical Vein Endothelial Cells.
  • protein renaturation means that denatured protein can restore its natural conformation and biological activity under appropriate conditions, and this phenomenon is called protein renaturation.
  • renaturation will restore the original spatial structure of the inactivated protein due to the change of the spatial structure and regain activity, but a protein does not necessarily have only one spatial structure.
  • endostatin the inventors found that although endostatin, which is insoluble under the condition of physiological pH value, becomes soluble through so-called "refolding", it also loses its activity at the same time, because the vascular endothelial The highly active state of inhibin is insoluble instead.
  • Endostar is a developed antineoplastic drug with human endostatin as the active ingredient.
  • Endu Recombinant human endostatin injection
  • Endu is a class 1.1 anti-tumor blood vessel targeting drug researched and developed by Chinese scientists on the basis of natural human endostatin with 9 amino acids added.
  • reduced human endostatin refers to human endostatin containing at most one pair (1 pair or 0 pair) of disulfide bonds.
  • reduced human endostatin there is no disulfide bond between Cys33 and Cys173 residues in the human endostatin molecule, or there is no disulfide bond between Cys135 and Cys165 residues. A disulfide bond is formed, or neither disulfide bond is formed.
  • Oxidized human endostatin refers to human endostatin comprising two pairs of disulfide bonds. Also take Figure 7 as an example, in “oxidized human endostatin", Cys33 of the human endostatin molecule forms a disulfide bond with the sulfhydryl group on the Cys173 residue, and the thiol group on the Cys135 and Cys165 residue also forms a disulfide bond. key.
  • reduced human endostatin analogs include cysteine mutations of natural endostatin, partial peptides of natural endostatin, changes in the amino acid sequence of natural endostatin, or the above The combination of the three conditions, as long as the condition of "containing at most one pair of disulfide bonds" is satisfied, but still retains the anti-angiogenesis and anti-tumor activities of the reduced human vascular endostatin.
  • the cysteine mutation of natural vascular endostatin refers to the deletion or substitution of one or more cysteines on the basis of the amino acid sequence of natural vascular endostatin; changing the amino acid sequence of natural vascular endostatin refers to the Based on the amino acid sequence of endostatin, a part of the amino acid sequence is inserted, deleted or substituted.
  • the reduced human endostatin analog contains at most one pair of disulfide bonds.
  • insoluble at physiological pH means that reduced human endostatin is insoluble in water at about pH 7.4, for example, insoluble in water at pH 7.35-7.45.
  • the reduced human endostatin of the present invention is insoluble in water under physiological pH conditions, but is soluble in water under other pH conditions, for example, it is soluble in water when the pH is less than 5.5.
  • Conductivity herein refers to the ability of a solution to conduct an electric current.
  • the conductivity of the solution can be changed by changing the salt concentration in the solution, eg, the conductivity of the elution buffer can be increased by increasing the salt concentration in the elution buffer.
  • Salts that can be used to increase conductivity include, but are not limited to, potassium chloride (KCl), sodium chloride (NaCl), potassium carbonate, sodium acetate, potassium sulfate, sodium sulfate, citrate, phosphate, or mixtures of these salts .
  • vascular endostatin It is a globular protein with two pairs of nested disulfide bonds, Cys33-Cys173 and Cys135-Cys165 (as shown in Figure 7); the inhibitory activity of endostatin on endothelial cell migration and proliferation may be related to The structures of disulfide bond retention are closely related, especially the disulfide bond Cys135-Cys165.
  • the inventors of the present invention unexpectedly found that the highly active structure of endostatin is reduced human endostatin, which contains at most one pair of disulfide bonds and is insoluble at physiological pH.
  • the present invention also proves that the activity of vascular endostatin is related to its cysteine state and protein space structure, but the present invention finds that the highly active structure of vascular endostatin is reduced human vascular endostatin, rather than the existing
  • the technique generally considers the oxidized state of human endostatin. Therefore, the reduced human endostatin of the present invention is not only different in structure from the oxidized human endostatin, but also has higher biological activity.
  • This highly active structure is not limited to reduced human endostatin containing cysteine residues in its molecule, but also includes reduced human endostatin analogs, as long as the "containing at most one pair of disulfide bonds "This condition is sufficient.
  • the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO: 1, 2, 3 or 4.
  • the reduced human endostatin analogs of the present invention include at least one or a combination of the following:
  • the reduced human endostatin analog contains at most one pair of disulfide bonds.
  • the reduced human endostatin analog molecule of the present invention may contain four cysteine residues, three cysteine residues, two cysteine residues, one cysteine residue or 0 cysteine residues, but no matter how many cysteine residues are present, at most one pair of disulfide bonds is formed.
  • replacement refers to the replacement of cysteine with other amino acids, and the substituted endostatin still has activity and better druggability.
  • the present invention does not limit the position of the cysteine that does not form a disulfide bond in the reduced human endostatin, and the position of the cysteine will vary according to the amino acid sequence of the reduced human endostatin.
  • the amino acid sequence of the reduced human vascular endostatin is as shown in SEQ ID NO: 1, wherein the formation of disulfide bonds includes:
  • Cys135 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys165 residue;
  • the reduced human endostatin of the present invention is directly purified from the inclusion body expressed by Escherichia coli.
  • inclusion bodies are high-density, insoluble protein particles wrapped by membranes formed when foreign genes are highly expressed in prokaryotic cells, especially Escherichia coli.
  • Biologically active proteins in cells often exist in the form of soluble proteins or molecular complexes, and functional proteins are always folded into a specific three-dimensional structure. Proteins in inclusion bodies are aggregates in an unfolded state and have no biological activity.
  • endostatin it is usually obtained by genetic engineering (for example, by using the expression system of Escherichia coli).
  • Recombinant human endostatin produced by Escherichia coli expression system has no natural folding structure, poor water solubility and easy to form precipitates.
  • the precipitated recombinant human endostatin is usually refolded (refolded).
  • Chinese patent 00107569.1 discloses: by modifying the nucleotide coding sequence of human endostatin, recombinant human endostatin with an additional amino acid sequence (MGGSHHHHH) at the N-terminal is produced (rhEndostatin, named: Endostar, Chinese name: Endo), and specifically disclosed the treatment process of inclusion bodies: the inclusion bodies expressed by Escherichia coli were dissolved in Tris buffer containing 7M guanidine hydrochloride and 50mM mercaptoethanol and continued to place After 1 hour, centrifuge to take the supernatant.
  • MGSHHHHH additional amino acid sequence
  • the supernatant was purified with a Ni 2+ column, and the eluted product containing endostatin protein eluted from the Ni 2+ column was rapidly diluted at a volume ratio of 1:10 in a solution containing 2.5M urea, 100mM NaCl, 0.1M Tris -HCl (pH8.0), 2mM reduced glutathione and 0.02mM oxidized glutathione solution to refold (refold) the protein.
  • free sulfhydryl groups on cysteines form disulfide bonds by adding, for example, oxidized glutathione.
  • the refolded recombinant human endostatin has two pairs of disulfide bonds, and is soluble in water under physiological pH conditions (eg, pH 7.4).
  • the reduced human endostatin in the present invention is directly purified from the inclusion body expressed by Escherichia coli, and its activity is much higher than that of the oxidized human endostatin. Therefore, the present invention provides a method for preparing the above-mentioned reduced human endostatin.
  • the vector containing the nucleotide sequence encoding human endostatin is transformed into a prokaryotic expression system (such as Escherichia coli), and the inclusion is obtained through fermentation and expression.
  • the inclusion body was dissolved and purified to obtain reduced human endostatin.
  • nucleotide sequence encoding human endostatin is the nucleus encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. nucleotide sequence.
  • SEQ ID NO: 1 and SEQ ID NO: 2 are identical with SEQ ID NO: 1, SEQ ID NO: 2 has one more methionine at the N-terminal, and SEQ ID NO: 3 and SEQ ID NO: 4 The difference is that there is one more methionine at the N-terminal of SEQ ID NO:4 compared with SEQ ID NO:3.
  • SEQ ID NO: 2 The difference between SEQ ID NO: 2 and SEQ ID NO: 4 is that the N-terminus of SEQ ID NO: 4 has an additional amino acid sequence (MGGSHHHHH), and SEQ ID NO: 4 is the amino acid sequence of the existing product Endu.
  • SEQ ID NO: 4 is the amino acid sequence of the existing product Endu.
  • Endostar The difference between the reduced recombinant human endostatin of the present invention and Endostar is that the reduced recombinant human endostatin of the present invention has not been refolded, and the inclusion bodies expressed by E. Purify it.
  • Amino acid sequence 1 (SEQ ID NO: 1):
  • Amino acid sequence 2 (SEQ ID NO: 2):
  • Amino acid sequence 3 (SEQ ID NO: 3):
  • Amino acid sequence 4 (SEQ ID NO:4):
  • the reduced human vascular endostatin or its analogues of the present invention have higher activity than the oxidized human endostatin, but they are insoluble in water at physiological pH value and cannot satisfy Druggability requirements.
  • the present invention attempts to use modifiers to modify the reduced human endostatin or its analogs.
  • the present invention modifies the reduced human endostatin or its analogs with modifiers, and while maintaining the highly active structure of the reduced human endostatin or its analogs, obtains good solubility under physiological pH value conditions.
  • the modification also increases the molecular weight of the drug, reduces the rate of renal filtration, and prolongs the half-life of the drug.
  • the present invention provides a modified reduced human vascular endostatin or its analogs, wherein the modification is selected from any one of high molecular polymers, protein molecules or fragments thereof, small molecular substances or a combination thereof .
  • Modification refers to the covalent modification or non-covalent combination of a modified substance (such as a high molecular polymer or a small molecular substance) or a non-covalent combination of a reduced human vascular endostatin or its analogues, or a modified substance (such as a protein molecule or a fragment thereof) combined with Reduced human endostatin or its analogs form a fusion protein.
  • protein molecules or fragments thereof include albumin, immunoglobulins, cytokines or fragments thereof, preferably Fc fragments of immunoglobulins.
  • the reduced human endostatin or its analogs modified by the immunoglobulin Fc fragment can be obtained through chemical modification or fusion expression.
  • the reduced human endostatin modified by the immunoglobulin Fc fragment is expressed by fusion, the reduced human endostatin modified by the immunoglobulin Fc fragment is the formation of the immunoglobulin Fc fragment and the reduced human endostatin fusion protein.
  • the present invention provides a modified reduced human vascular endostatin or its analogues, which can maintain a high activity state and be soluble in water under physiological pH value conditions.
  • the soluble reduced human Endostatin or its analogues can be used to measure cell viability in vitro and make it possible to further develop drugs for systemic administration.
  • the reduced human endostatin or its analogues are modified with a polymer. More preferably, the reduced human endostatin or its analogs are modified with polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Polyethylene glycol molecules are amphiphilic and can be dissolved in both water and most organic solvents. They are non-toxic, non-immunogenic, and highly soluble in aqueous solutions. Coupling proteins with hydrophilic polymers such as polyethylene glycol can increase protein stability, reduce non-specific adsorption and immunogenicity. When the conjugate reaches a certain molecular weight, it can greatly reduce the elimination efficiency of the kidney, and is an effective method to prolong the half-life of protein drugs in vivo.
  • the initial polyethylene glycol modification used amino groups as reactive sites, mainly including the N-terminal ⁇ -amino group of the protein and the ⁇ -amino group on the side chain of the lysine residue.
  • the product of this type of reaction is the coupling of a protein molecule to one or more polyethylene glycol molecules.
  • the modification of the ⁇ -amino group on the side chain of lysine residues often produces modified isomers because the reaction site is not specific.
  • WO 2007082483A1 discloses PEG-modified endostatin (Endostatin), but the endostatin used in this document is the endostatin (oxidized endostatin) after refolding Human vascular endostatin), containing two pairs of disulfide bonds, is soluble in water at physiological pH. The basis for this inference is as follows:
  • the N-terminally modified recombinant human endostatin product has an inhibitory rate to the migration of endothelial cells in an in vitro cytology experiment that is lower than that of the unmodified protein. 2 times or more, the biological activity of the protein has been greatly improved, and this phenomenon of significantly increased in vitro activity has not been reported.”
  • Vascular endostatin used in cell experiments must be soluble under physiological pH conditions, otherwise Cannot be used to measure activity. Therefore, the above statement shows that the endostatin used in this document is in a soluble state at physiological pH, that is, the so-called refolding state with two pairs of disulfide bonds.
  • the preparation method of endostatin in this document is that the expression of inclusion bodies in Escherichia coli is renatured in vitro, and some endostatin has not formed disulfide bonds or has incomplete disulfide bond pairing, because polyethylene glycol
  • the modification makes this part of endostatin in the reduced state remain, instead of forming a precipitate as usual, and the increase in its activity is caused by this part of unrefolded endostatin.
  • the reduced human endostatin modified with polyethylene glycol has not been disclosed in the prior art.
  • the inventor compared the activity of polyethylene glycol-modified reduced human endostatin with polyethylene glycol-modified Endostar (oxidized human endostatin), and the experimental results proved that 1, 2, and 5 ⁇ g/ml
  • the inhibitory rates of polyethylene glycol-modified reduced recombinant human endostatin (nES) to inhibit the migration of HMEC cells were 28%, 76% and 90%, respectively, and the administration concentration was 10 ⁇ g/ml Endostar
  • the inhibition rate of inhibiting HMEC cell migration was 48%, and the inhibition rate of HMEC cell migration inhibited by polyethylene glycol-modified Endostar at a concentration of 10 ⁇ g/ml was 54%.
  • the embodiments of the present invention have proved that the reduced human endostatin modified with polyethylene glycol has higher activity than the oxidized human endostatin modified with polyethylene glycol, and the activity of the former is higher than that of the latter. 5-10 times.
  • the examples of the present invention further prove that the highly active polyethylene glycol-modified reduced human endostatin can be further purified, and the activity of specific components obtained after ion-exchange purification will continue to increase by about 10 times. That is, the activity of this component is 50-100 times that of the oxidized human endostatin modified with polyethylene glycol (see Example 2).
  • the activated polyethylene glycol is mixed with reduced human endostatin or its analogs and reacted to obtain modified reduced human endostatin or its analogs.
  • monomethoxypolyethylene glycol propionaldehyde is mixed with reduced human endostatin, and the reducing agent sodium cyanoborohydride is added for reaction; in the presence of sodium cyanoborohydride, monomethoxy Reductive amination reaction occurs between polyethylene glycol propionaldehyde and the primary amine of the N-terminal amino bond of reduced human endostatin.
  • the ratio of monomethoxy polyethylene glycol propionaldehyde to reduced human endostatin is 1:1, the pH value of the reaction is 4.5-5.5, and the reaction time is 4-6 hours.
  • the reaction temperature is room temperature.
  • the reduced human endostatin in a highly active state is further enriched by purification, taking the in vitro activity as an indication. Therefore, the above preparation method also includes purifying the coupling product of polyethylene glycol and reduced human endostatin by cation exchange chromatography (the medium is sulfopropyl sephadex (abbreviated as SP)).
  • the medium is sulfopropyl sephadex (abbreviated as SP)) to obtain specific components (for example, components eluted with an elution buffer with a conductivity of 25 mS/cm)
  • specific components for example, components eluted with an elution buffer with a conductivity of 25 mS/cm
  • the activity is 50-100 times that of the oxidized human endostatin modified by polyethylene glycol.
  • the present invention provides a pharmaceutical composition, which comprises the above-mentioned reduced human endostatin or its analogue or the above-mentioned modified reduced human endostatin or its analogue, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises an effective amount (for example, 0.01-99.9% by weight) of reduced human endostatin or its analogs or modified reduced human endostatin or its analogs, and a pharmaceutically acceptable carrier .
  • Said carrier is for example, but not limited to, diluents (such as water), excipients, etc.; binders, such as cellulose derivatives, gelatin, polyvinylpyrrolidone, etc.; fillers such as starch, etc.; disintegrants such as calcium carbonate, carbonic acid sodium hydrogen; lubricants such as calcium stearate or magnesium stearate, etc.
  • diluents such as water
  • excipients such as water
  • binders such as cellulose derivatives, gelatin, polyvinylpyrrolidone, etc.
  • fillers such as starch, etc.
  • disintegrants such as calcium carbonate, carbonic acid sodium hydrogen
  • lubricants such as calcium stearate or magnesium stearate, etc.
  • other adjuvants such as flavoring and sweetening agents can also be added to the composition.
  • When used for oral administration it can be prepared into conventional solid preparations such as tablets, powders or capsules, etc.; when used
  • the pharmaceutical composition of the present invention can be made into common preparations or sustained-release preparations.
  • the sustained-release preparation is selected from microcapsules, hydrogels, microspheres, miniature osmotic pumps or liposomes and the like. Making the above-mentioned reduced human endostatin or its analogs or the above-mentioned modified reduced human endostatin or its analogs into sustained-release preparations can prolong the half-life of reduced human endostatin or its analogs in vivo .
  • the above-mentioned reduced human endostatin or its analogues or modified reduced human endostatin or its analogues are put into a pharmaceutical carrier (such as hydrogel, liposome, etc.), so that The reduced human endostatin or its analogue is slowly released from the pharmaceutical carrier, and a stable concentration of the reduced human endostatin or its analogue is maintained in the body.
  • a pharmaceutical carrier such as hydrogel, liposome, etc.
  • reduced human endostatin or its analogs and polyethylene glycol-modified reduced human endostatin or its analogs have the activity of inhibiting the proliferation and migration of vascular endothelial cells. Therefore, the reduced human endostatin or its analogue, the polyethylene glycol modified reduced human endostatin or its analogue and the corresponding pharmaceutical composition of the present invention can be used to prepare antitumor drugs.
  • the tumor is selected from lung cancer, neuroendocrine tumor, colon cancer, bone cancer, liver cancer, gastric cancer, pancreatic cancer, oral cancer, breast cancer, prostate cancer, lymphatic cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, cervical cancer Carcinoma, sarcoma, kidney cancer, gallbladder cancer and malignant melanoma.
  • the reduced human endostatin or its analogs and polyethylene glycol-modified reduced human endostatin or its analogs of the present invention, and the corresponding pharmaceutical composition can treat other diseases related to angiogenesis, such as macular degeneration. Therefore, the reduced human endostatin or its analogue, polyethylene glycol-modified reduced human endostatin or its analogue and the corresponding pharmaceutical composition of the present invention can be used to prepare anti-angiogenesis drugs.
  • the present invention proposes a new point of view, that is, insoluble proteins (such as transmembrane proteins, collagen and fibronectin, etc.) which account for a large proportion of proteins in the body play important biological functions locally. Poor solubility under certain conditions, its structure and function are difficult to directly study with the existing technology, and it is also difficult to develop into a drug for systemic administration. It is more convenient to increase the solubility of this type of protein under the condition of physiological pH value through modification. Explore its structure and function, and improve its druggability.
  • insoluble proteins such as transmembrane proteins, collagen and fibronectin, etc.
  • the present invention provides a modified protein or polypeptide, wherein the protein or polypeptide is insoluble in water under physiological pH conditions, and the modification is selected from high molecular polymers, protein molecules or fragments thereof, small molecular substances any one or combination of them.
  • modifiers such as high molecular polymers or small molecular substances
  • modifiers can be used to covalently modify or non-covalently bind proteins or polypeptides that are insoluble in water under physiological pH conditions, or modifiers (such as protein molecules or Fragments thereof) form fusion proteins with proteins or polypeptides that are insoluble in water under physiological pH conditions.
  • protein molecules or fragments thereof include albumin, immunoglobulins, cytokines or fragments thereof, etc., preferably immunoglobulin Fc fragments, proteins or polypeptides modified by immunoglobulin Fc fragments ( Insoluble in water under the condition of physiological pH value) can be obtained by chemical modification, and can also be obtained by fusion expression.
  • the protein or polypeptide modified by the Fc fragment of immunoglobulin is expressed by fusion
  • the protein or polypeptide modified by Fc fragment of immunoglobulin is the fusion protein formed by the Fc fragment of immunoglobulin and the protein or polypeptide.
  • the protein or polypeptide that is insoluble in water under physiological pH value conditions is modified with a high molecular polymer. More preferably, the protein or polypeptide that is insoluble in water at physiological pH is modified with polyethylene glycol.
  • polyethylene glycol is usually used to increase the half-life of drug metabolism in vivo.
  • the present invention utilizes polyethylene glycol to modify the water - insoluble protein or polypeptide under the physiological pH value condition, which can increase the solubility of the water-insoluble protein or polypeptide under the physiological pH value condition, so that it becomes less soluble under the physiological pH value condition.
  • the protein or polypeptide insoluble in water under the condition of physiological pH value of the present invention includes but not limited to natural protein or polypeptide or analogues thereof.
  • endostatin from animal sources has similar functions to endostatin from human sources and can be used to develop similar drugs.
  • the homology of endostatin from different species is as follows:
  • the present invention provides the use of animal-derived vascular endostatin in the preparation of veterinary medicine.
  • the veterinary medicine can be used to treat cancers in animals, such as liver cancer, esophageal cancer, gastric cancer, breast cancer, lymphoma, and malignant melanoma; the veterinary medicine can also be used to treat diseases related to angiogenesis in animals, such as macular degeneration.
  • Comparative example 1 polyethylene glycol modified endostar
  • Endostar used in this comparative example is produced by Shandong Xiansheng Medzin Biopharmaceutical Co., Ltd. and is commercially available. Dialyze Endostar into 30mM HAc-NaAc, pH4.5-5.5 buffer solution, add monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD, 20kDa) in equivalent amount, and add reducing agent cyanide at a final concentration of 20mM Sodium borohydride (NaBH 3 CN), stirred evenly, left at room temperature for 4-6 hours, and detected by SDS-PAGE electrophoresis. It was confirmed by SDS-PAGE electrophoresis that the polyethylene glycol-modified endostar had been successfully obtained.
  • mPEG-ALD monomethoxypolyethylene glycol propionaldehyde
  • NaBH 3 CN Sodium borohydride
  • Embodiment 1 polyethylene glycol modified recombinant human endostatin in reduced state
  • the reduced recombinant human endostatin involved in this embodiment includes recombinant human endostatin with "amino acid sequence 2" or "amino acid sequence 4".
  • the DNA fragment containing the gene encoding "amino acid sequence 1" or "amino acid sequence 3" was cloned into Escherichia coli expression vector pET30a, the constructed vector was transformed into Escherichia coli, and the target protein expressed by fermentation was an inclusion body (the protein expressed in Escherichia coli would be in The N-terminus automatically adds methionine).
  • the inclusion bodies expressed by Escherichia coli were dissolved in 20mM Tris-HCl buffer solution containing 7M guanidine hydrochloride or 8M urea and 20mM dithiothreitol (DTT), left at room temperature for 3-6 hours, centrifuged to get the supernatant, from Purification of reduced recombinant human endostatin from the supernatant.
  • DTT dithiothreitol
  • the purified reduced recombinant human endostatin was dialyzed into 30mM HAc-NaAc, pH 4.5-5.5 buffer, and an equivalent amount of monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD, 20kDa) was added , add a final concentration of 20mM sodium cyanoborohydride (NaBH 3 CN), stir evenly, let stand at room temperature for 4-6 hours, and detect by SDS-PAGE electrophoresis ( Figure 1).
  • the reduced recombinant human endostatin involved in this embodiment includes recombinant human endostatin having "amino acid sequence 2".
  • the preparation method of the reduced recombinant human vascular endostatin in this example is the same as that in Example 1.
  • the reduced recombinant human endostatin was dialyzed into 30mM HAc-NaAc, pH4.5-5.5 buffer solution, and the equivalent amount of monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD, 20kDa) was added, and the final Concentration of 20mM reducing agent sodium cyanoborohydride (NaBH3CN), stirring evenly, standing at room temperature for 4-6 hours, SDS-PAGE electrophoresis detection.
  • mPEG-ALD monomethoxypolyethylene glycol propionaldehyde
  • a polyethylene glycol is coupled to a reduced endostatin molecule, and the coupling site is the ⁇ -amino group at the N-terminal of the reduced endostatin, a small amount of reduced endostatin will be non-specifically multipositioned Point decorated or undecorated.
  • the reaction solution can be directly used for column purification to remove multi-modified and unmodified reduced endostatin.
  • SP sulfopropyl sephadex
  • the 25mS/cm component is the component eluted with an elution buffer with a conductivity of 25mS/cm.
  • the 30mS/cm group Fractions are fractions eluted with an elution buffer with a conductivity of 30mS/cm, and fractions with a conductivity of 40mS/cm are fractions eluted with an elution buffer with a conductivity of 40mS/cm.
  • Test Example 1 Determination of the inhibitory activity of polyethylene glycol-modified reduced recombinant human endostatin on HMEC cell migration
  • HMEC cells were used to determine the inhibitory activity of the polyethylene glycol-modified endostar and polyethylene glycol-modified reduced recombinant human endostatin prepared in Comparative Example 1 and Example 1 on HMEC cell migration.
  • HMEC cells in the logarithmic growth phase take a 24-well plate, first add 800 ⁇ l of cell culture medium to each well, and then add 200 ⁇ l of drugs with different concentrations to each well, and the final volume is 1 ml. Take another 24-well plate and place the Transwell chamber on the well. The HMEC cells were digested with 0.25% trypsin-EDTA, centrifuged, resuspended in culture medium and counted, and the cell concentration was adjusted to 10 6 cells/ml. Take 160 ⁇ l of cell suspension and add it to the upper chamber of the Transwell, and add 40 ⁇ l of different concentrations of drugs. After incubation, the Transwell chamber is placed in a 24-well plate filled with drugs. Place the 24-well plate in a 37 °C, 5% CO2 incubator for 4 h. The upper chamber of the Transwell was removed, and the cell migration in the 24-well plate was detected by fluorescence method.
  • the positive control 10 ⁇ g/ml Endostar (Endostar) inhibition rate was 48%
  • the polyethylene glycol modified Endostar (PEG-Endostar, prepared in Comparative Example 1) inhibition rate of 10 ⁇ g/ml was 54%
  • the blank control is a buffer solution (30mM HAc-NaAc, pH5.2 Buffer)
  • the inhibition rate of each administration group is calculated on the basis of the blank control
  • the inhibition rate of the blank control group is assumed when calculating the inhibition rate of each administration group is 0 ( Figure 2).
  • Test example 2 measures the inhibitory activity of each component of purification to HMEC cell migration
  • HMEC cells were used to measure the inhibitory activity of FT (effluent), 25, 30, 40 mS/cm components prepared in Example 3 and polyethylene glycol-modified Endostar prepared in Comparative Example 1 on HMEC cell migration.
  • HMEC cells in the logarithmic growth phase take a 24-well plate, first add 800 ⁇ l of cell culture medium to each well, and then add 200 ⁇ l of drugs with different concentrations to each well, and the final volume is 1 ml. Take another 24-well plate and place the Transwell chamber on the well. The HMEC cells were digested with 0.25% trypsin-EDTA, centrifuged, resuspended in culture medium and counted, and the cell concentration was adjusted to 10 6 cells/ml. Take 160 ⁇ l of cell suspension and add it to the upper chamber of the Transwell, and add 40 ⁇ l of different concentrations of drugs. After incubation, the Transwell chamber is placed in a 24-well plate filled with drugs. Place the 24-well plate in a 37 °C, 5% CO2 incubator for 4 h. The upper chamber of the Transwell was removed, and the cell migration in the 24-well plate was detected by fluorescence method.
  • the inhibition rate of FT (effluent), 25, 30, 40mS/cm component is respectively-6%, 94%, 80%, 15%
  • the endostar (Endostar) inhibition rate of positive control 10 ⁇ g/ml is 53%
  • the Endostar (PEG-Endostar, prepared in comparative example 1) inhibition rate of 10 ⁇ g/ml polyethylene glycol modification is 58%
  • blank control is buffer solution (30mM HAc-NaAc, pH5.2)
  • the inhibition rate of each administration group is calculated on the basis of the blank control
  • the inhibition rate of the blank control group is assumed to be 0 (Fig. 3) when calculating the inhibition rate of each administration group. It can be seen from Figure 3 that the 25mS/cm component has the highest activity.
  • the 25mS/cm components at 0.1, 0.2, 0.5, and 1.0 ⁇ g/ml concentration were measured to inhibit the migration of HMEC cells, and the inhibition rates were 35%, 73%, 81%, and 92%, respectively, and the positive control was 10 ⁇ g/ml Endostar (Endostar) inhibition rate is 43%, 10 ⁇ g/ml polyethylene glycol modified Endostar (PEG-Endostar, prepared in comparative example 1) inhibition rate is 41%, blank control is buffer solution (30mM HAc-NaAc , pH5.2Buffer), the inhibition rate of each administration group was calculated on the basis of the blank control, and the inhibition rate of the blank control group was assumed to be 0 (Fig.
  • Test Example 3 Determination of the inhibitory activity of polyethylene glycol-modified reduced recombinant human endostatin on HMEC or HUVEC cell proliferation
  • HUVEC cells were cultured and subcultured to passage 3-5, and the cells were ready to be inoculated when the cells were in good condition.
  • the reduced recombinant human endostatin modified with polyethylene glycol was diluted with 20 mM PB buffer solution, three parallel wells were set up for each gradient, and 40 ⁇ l was added to each well of a 96-well plate.
  • the cell density is 6000 cells/ml, add 160 ⁇ l cell suspension to each well of the previous 96-well culture plate, culture at 37°C, 5% CO 2 incubator for 48-72h, and measure the proliferation activity by MTT method.

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Abstract

The present invention relates to a reduction-state human endostatin or an analog thereof, and a modified reduction-state human endostatin or an analog thereof. Compared to the existing oxidation-state human endostatin, the reduction-state human endostatin or the analog thereof and the modified reduction-state human endostatin or the analog thereof have better anti-angiogenic and anti-tumor activities.

Description

一种抗血管生成药物an antiangiogenic drug 技术领域technical field
本发明涉及生物医药技术领域，尤其涉及一种还原态人血管内皮抑制素或其类似物和修饰的还原态人血管内皮抑制素或其类似物。本发明还涉及一种修饰的蛋白或多肽，通过修饰使生理pH值条件下不可溶于水的蛋白或多肽变得可溶于水，从而提高其成药性。The invention relates to the technical field of biomedicine, in particular to a reduced human endostatin or an analog thereof and a modified reduced human endostatin or an analog thereof. The present invention also relates to a modified protein or polypeptide, which can be modified to make the water-insoluble protein or polypeptide water-soluble under the condition of physiological pH value, thereby improving its druggability.
背景技术Background technique
血管内皮抑制素(Endostatin)是胶原XVIII羧基端的一段分子量大小为20kDa的酶切产物。1997年美国哈佛大学Judah Folkman教授等人在血管内皮瘤细胞的培养物中发现此蛋白，它具有抑制血管内皮细胞增殖、迁移和体内血管生成的活性(O’Reilly MS,Boehm T,Shing Y,Fukai N,Vasios G,Lane WS,Flynn E,Birkhead JR,Olsen BR,Folkman J:Endostatin:an endogenous inhibitor of angiogenesis and tumor growth.Cell 88:277–85,1997)。Judah Folkman教授等人的进一步研究发现：重组血管内皮抑制素可以抑制小鼠体内多种肿瘤的生长、转移，甚至能完全治愈肿瘤，而且不产生耐药性；其发挥活性的机理在于它通过抑制血管内皮细胞的生长，抑制了肿瘤组织附近的新生血管的生成，使得肿瘤组织得不到生长所必需的大量营养和氧气，最后停止生长或坏死(Boehm T,Folkman J,Browder T,O’Reilly MS:Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance.Nature 390:404–7,1997)。Endostatin is an enzyme-cleaved product with a molecular weight of 20 kDa at the carboxy-terminal of collagen XVIII. In 1997, Professor Judah Folkman of Harvard University and others discovered this protein in the culture of hemangioendothelioma cells, which has the activity of inhibiting the proliferation, migration and angiogenesis of vascular endothelial cells (O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J: Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88:277–85, 1997). Further research by Professor Judah Folkman and others found that recombinant endostatin can inhibit the growth and metastasis of various tumors in mice, and even completely cure tumors without producing drug resistance; the mechanism of its activity is that it inhibits The growth of vascular endothelial cells inhibits the formation of new blood vessels near the tumor tissue, so that the tumor tissue cannot obtain the necessary nutrients and oxygen for growth, and finally stops growth or necrosis (Boehm T, Folkman J, Browder T, O'Reilly MS: Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390:404–7, 1997).
通过基因工程制备的重组人血管内皮抑制素可以作为肿瘤治疗药物。目前唯一获批上市的重组人血管内皮抑制素药物商品名为“恩度(Endostar)”。最初的研究发现血管内皮抑制素有极高的抗血管生成活性和抗肿瘤活性，但后续的基础研究和临床研究显示的活性并不理想(B.Kim Lee Sim,Nicholas J.MacDonald and Edward R.Gubish：Angiostatin and Endostatin:Endogenous Inhibitors of Tumor Growth.Cancer and Metastasis Reviews 19:181–190,2000)。因此，在实际应用中，需要抗肿 瘤活性更好的血管内皮抑制素。The recombinant human endostatin prepared by genetic engineering can be used as a tumor treatment drug. The trade name of the only recombinant human endostatin drug approved for marketing at present is "Endostar". The initial research found that endostatin has extremely high anti-angiogenic activity and anti-tumor activity, but the subsequent basic research and clinical research showed that the activity was not ideal (B.Kim Lee Sim, Nicholas J.MacDonald and Edward R. Gubish: Angiostatin and Endostatin: Endogenous Inhibitors of Tumor Growth. Cancer and Metastasis Reviews 19:181–190, 2000). Therefore, in practical applications, endostatins with better antitumor activity are needed.
发明内容Contents of the invention
有鉴于此，本发明的目的是提供一种还原态人血管内皮抑制素或其类似物和修饰的还原态人血管内皮抑制素或其类似物，该还原态人血管内皮抑制素或其类似物和修饰的还原态人血管内皮抑制素或其类似物相对于现有的氧化态人血管内皮抑制素具有更好的抗血管生成和抗肿瘤活性。In view of this, the purpose of the present invention is to provide a reduced human endostatin or its analog and modified reduced human endostatin or its analog, the reduced human endostatin or its analog Compared with the existing oxidized human endostatin, the modified reduced human endostatin or its analogues have better anti-angiogenesis and anti-tumor activities.
基于上述目的，本发明的第一个方面提供了一种还原态人血管内皮抑制素或其类似物，所述还原态人血管内皮抑制素包含至多一对二硫键，所述二硫键由人血管内皮抑制素分子的两个半胱氨酸残基上的巯基形成。Based on the above purpose, the first aspect of the present invention provides a reduced state of human endostatin or its analogs, the reduced state of human endostatin contains at most a pair of disulfide bonds, the disulfide bond consists of Sulfhydryl formation on two cysteine residues of the human endostatin molecule.
在本发明的优选的实施方案中，所述还原态人血管内皮抑制素类似物包括以下的至少一种或其组合：In a preferred embodiment of the present invention, the reduced human endostatin analogs include at least one or a combination of the following:
---在天然血管内皮抑制素的氨基酸序列基础上一个或多个半胱氨酸的缺失或置换；--- Deletion or substitution of one or more cysteines on the basis of the amino acid sequence of natural vascular endostatin;
---天然血管内皮抑制素的部分肽段；或者--- Partial peptides of natural endostatin; or
---改变天然血管内皮抑制素的氨基酸序列；---Change the amino acid sequence of natural vascular endostatin;
其中，所述还原态人血管内皮抑制素类似物包含至多一对二硫键。Wherein, the reduced human endostatin analog contains at most one pair of disulfide bonds.
在本发明的优选的实施方案中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:1、2、3或4所示。In a preferred embodiment of the present invention, the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO: 1, 2, 3 or 4.
在本发明的优选的实施方案中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:1所示，其中二硫键形成情况包括：In a preferred embodiment of the present invention, the amino acid sequence of the reduced human endostatin is as shown in SEQ ID NO: 1, wherein the formation of disulfide bonds includes:
---还原态人血管内皮抑制素分子中的Cys33与Cys173残基上的巯基没有形成二硫键；---Cys33 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys173 residue;
---还原态人血管内皮抑制素分子中的Cys135与Cys165残基上的巯基没有形成二硫键；或者--- Cys135 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys165 residue; or
---以上两个二硫键都没有形成。---The above two disulfide bonds are not formed.
本发明第二方面还提供了一种修饰的还原态人血管内皮抑制素或其类似物，所述还原态人血管内皮抑制素包含至多一对二硫键，所述二硫键由人血管内皮抑制素分子的两个半胱氨酸残基上的巯基形成。The second aspect of the present invention also provides a modified reduced human endostatin or its analogs, the reduced human endostatin contains at most one pair of disulfide bonds, and the disulfide bonds are formed by human vascular endothelial Sulfhydryl groups formed on two cysteine residues of the inhibin molecule.
在本发明的优选的实施方案中，所述还原态人血管内皮抑制素类似物包括以下的至少一种或其组合：In a preferred embodiment of the present invention, the reduced human endostatin analogs include at least one or a combination of the following:
---在天然血管内皮抑制素的氨基酸序列基础上一个或多个半胱氨酸的缺失或置换；--- Deletion or substitution of one or more cysteines on the basis of the amino acid sequence of natural vascular endostatin;
---天然血管内皮抑制素的部分肽段；或者--- Partial peptides of natural endostatin; or
---改变天然血管内皮抑制素的氨基酸序列；---Change the amino acid sequence of natural vascular endostatin;
其中，所述还原态人血管内皮抑制素类似物包含至多一对二硫键。Wherein, the reduced human endostatin analog contains at most one pair of disulfide bonds.
在本发明的优选的实施方案中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:1、2、3或4所示。In a preferred embodiment of the present invention, the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO: 1, 2, 3 or 4.
在本发明的优选的实施方案中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:2或4所示。In a preferred embodiment of the present invention, the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO: 2 or 4.
在本发明的优选的实施方案中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:1所示，其中二硫键形成情况包括：In a preferred embodiment of the present invention, the amino acid sequence of the reduced human endostatin is as shown in SEQ ID NO: 1, wherein the formation of disulfide bonds includes:
---还原态人血管内皮抑制素分子中的Cys33与Cys173残基上的巯基没有形成二硫键；---Cys33 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys173 residue;
---还原态人血管内皮抑制素分子中的Cys135与Cys165残基上的巯基没有形成二硫键；或者--- Cys135 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys165 residue; or
---以上两个二硫键都没有形成。---The above two disulfide bonds are not formed.
在本发明的优选的实施方案中，修饰物选自高分子聚合物、蛋白质分子或其片段、小分子物质中的任一种或其组合。In a preferred embodiment of the present invention, the modification is selected from any one or combination of high molecular polymers, protein molecules or fragments thereof, small molecular substances.
在本发明的优选的实施方案中，蛋白质分子或其片段包括白蛋白、免疫球蛋白、细胞因子或它们的片段等，优选为免疫球蛋白Fc片段。免疫球蛋白Fc片段修饰的还原态人血管内皮抑制素或其类似物可通过化学修饰得到，也可通过融合表达得到。当免疫球蛋白Fc片段修饰的还原态人血管内皮抑制素通过融合表达时，免疫球蛋白Fc片段修饰的还原态人血管内皮抑制素即为免疫球蛋白Fc片段与还原态人血管内皮抑制素形成的融合蛋白。In a preferred embodiment of the present invention, protein molecules or fragments thereof include albumin, immunoglobulins, cytokines or fragments thereof, preferably Fc fragments of immunoglobulins. The reduced human endostatin or its analogs modified by the immunoglobulin Fc fragment can be obtained through chemical modification or fusion expression. When the reduced human endostatin modified by the immunoglobulin Fc fragment is expressed by fusion, the reduced human endostatin modified by the immunoglobulin Fc fragment is the formation of the immunoglobulin Fc fragment and the reduced human endostatin fusion protein.
在本发明的优选的实施方案中，所述高分子聚合物为聚乙二醇。In a preferred embodiment of the present invention, the high molecular polymer is polyethylene glycol.
在本发明的优选的实施方案中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:2或4所示。In a preferred embodiment of the present invention, the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO: 2 or 4.
在本发明的优选的实施方案中，一个还原态人血管内皮抑制素或其类似物分子和一个或多个聚乙二醇分子偶联。In a preferred embodiment of the present invention, one reduced human endostatin or its analogue molecule is coupled to one or more polyethylene glycol molecules.
在本发明的优选的实施方案中，所述还原态人血管内皮抑制素或其类 似物与所述聚乙二醇偶联的位点是所述还原态人血管内皮抑制素或其类似物的N端α-氨基、赖氨酸残基侧链的ε-氨基、半胱氨酸残基侧链的巯基、天冬氨酸残基侧链的羧基、谷氨酸残基侧链的羧基中的一种或是他们的组合。In a preferred embodiment of the present invention, the site where the reduced human endostatin or its analogues are coupled to the polyethylene glycol is the site of the reduced human endostatin or its analogues. N-terminal α-amino group, ε-amino group of side chain of lysine residue, sulfhydryl group of side chain of cysteine residue, carboxyl group of side chain of aspartic acid residue, carboxyl group of side chain of glutamic acid residue one or a combination of them.
在本发明的优选的实施方案中，所述还原态人血管内皮抑制素或其类似物与所述聚乙二醇偶联的位点是所述还原态人血管内皮抑制素或其类似物N端的α-氨基。In a preferred embodiment of the present invention, the site where the reduced human endostatin or its analogs are coupled to the polyethylene glycol is the reduced human endostatin or its analogs N terminal α-amino group.
在本发明的优选的实施方案中，所述聚乙二醇分子的平均分子量在1,000到100,000道尔顿之间。In a preferred embodiment of the invention, said polyethylene glycol molecules have an average molecular weight between 1,000 and 100,000 Daltons.
在本发明的优选的实施方案中，所述聚乙二醇分子的平均分子量在5,000到40,000道尔顿之间。In a preferred embodiment of the invention, said polyethylene glycol molecules have an average molecular weight between 5,000 and 40,000 Daltons.
本发明的第三个方面提供了一种药物组合物，其包含上述还原态人血管内皮抑制素或其类似物或上述修饰的还原态人血管内皮抑制素或其类似物，以及药学上可接受的载体。The third aspect of the present invention provides a pharmaceutical composition, which comprises the above-mentioned reduced human endostatin or its analogue or the above-mentioned modified reduced human endostatin or its analogue, and pharmaceutically acceptable Carrier.
在本发明的优选的实施方案中，所述药物组合物为缓释制剂。In a preferred embodiment of the present invention, the pharmaceutical composition is a sustained-release preparation.
本发明的第四个方面提供了一种修饰的蛋白或多肽，所述蛋白或多肽在生理pH值条件下不可溶于水，修饰后其在生理pH值条件下可溶于水；修饰物选自高分子聚合物、蛋白质分子或其片段、小分子物质中的任一种或其组合。The fourth aspect of the present invention provides a modified protein or polypeptide, the protein or polypeptide is insoluble in water under the condition of physiological pH value, and after modification, it is soluble in water under the condition of physiological pH value; the modification is selected from Any one or a combination of high molecular weight polymers, protein molecules or their fragments, small molecular substances.
本发明的第五个方面提供了动物源血管内皮抑制素在制备兽药中的用途。The fifth aspect of the present invention provides the use of animal-derived vascular endostatin in the preparation of veterinary medicine.
从上面所述可以看出，本发明提供的一种还原态人血管内皮抑制素或其类似物和修饰的还原态人血管内皮抑制素或其类似物相对于现有的氧化态人血管内皮抑制素具有更好的抗血管生成和抗肿瘤活性。As can be seen from the above, a reduced human endostatin or its analogs provided by the present invention and modified reduced human endostatin or its analogs inhibit It has better anti-angiogenic and anti-tumor activities.
附图说明Description of drawings
图1为SDS-PAGE电泳检测还原态重组人血管内皮抑制素和聚乙二醇(PEG)修饰的还原态重组人血管内皮抑制素的电泳图；其中，L1和L2为聚乙二醇(PEG)修饰的还原态重组人血管内皮抑制素，L3和L4为还原态重组人血管内皮抑制素。Fig. 1 is the electrophoresis figure of the reduced recombinant human endostatin of reducing state detected by SDS-PAGE electrophoresis and the reduced recombinant human endostatin of polyethylene glycol (PEG); Wherein, L1 and L2 are polyethylene glycol (PEG) ) modified reduced recombinant human endostatin, L3 and L4 are reduced recombinant human endostatin.
图2为聚乙二醇修饰的还原态重组人血管内皮抑制素、恩度 (Endostar)和聚乙二醇修饰的恩度(Endostar)抑制HMEC细胞(人微血管内皮细胞)迁移的活性的对比图；其中，B为空白，L1为给药浓度为10μg/ml的恩度抑制HMEC细胞迁移的活性，L2为给药浓度为10μg/ml的聚乙二醇修饰的恩度(PEG-Endostar)抑制HMEC细胞迁移的活性，L3为给药浓度为1μg/ml的聚乙二醇修饰的还原态重组人血管内皮抑制素(nES)抑制HMEC细胞迁移的活性，L4为给药浓度为2μg/ml的聚乙二醇修饰的还原态重组人血管内皮抑制素(nES)抑制HMEC细胞迁移的活性，L5为给药浓度为5μg/ml的聚乙二醇修饰的还原态重组人血管内皮抑制素(nES)抑制HMEC细胞迁移的活性。Fig. 2 is the comparison chart of the activity that the reduction state recombinant human endostatin of PEG modification, endostar (Endostar) and the endostar (Endostar) of PEG modification inhibit HMEC cell (human microvascular endothelial cell) migration Wherein, B is blank, L1 is that the endostar that administration concentration is 10 μ g/ml inhibits the activity of HMEC cell migration, and L2 is that the endostar (PEG-Endostar) that administration concentration is 10 μ g/ml is modified inhibits The activity of HMEC cell migration, L3 is the activity that the reduced state recombinant human endostatin (nES) modified by polyethylene glycol with the administration concentration of 1 μg/ml inhibits the activity of HMEC cell migration, and L4 is the activity of the administration concentration of 2 μg/ml The reduced state recombinant human endostatin (nES) modified by polyethylene glycol inhibits the activity of HMEC cell migration, and L5 is the reduced recombinant human endostatin (nES) modified by polyethylene glycol with an administration concentration of 5 μg/ml ) inhibits the activity of HMEC cell migration.
图3为用阳离子交换层析(介质为磺酸基丙基葡聚糖凝胶(简称SP))纯化聚乙二醇修饰的还原态重组人血管内皮抑制素所得的各组分、恩度和聚乙二醇修饰的恩度抑制HMEC细胞迁移的活性的对比图；其中，B为空白，L1为给药浓度为10μg/ml的恩度抑制HMEC细胞迁移的活性，L2为给药浓度为10μg/ml的聚乙二醇修饰的恩度抑制HMEC细胞迁移的活性，L3为nES(FT(流出液))抑制HMEC细胞迁移的活性，L4为给药浓度为2ug/ml的nES(25mS/cm)组分抑制HMEC细胞迁移的活性，L5为给药浓度为2μg/ml的nES(30mS/cm)组分抑制HMEC细胞迁移的活性，L6为给药浓度为2μg/ml的nES(40mS/cm)组分抑制HMEC细胞迁移的活性。Fig. 3 is each component, endostar and endostatin obtained by purifying polyethylene glycol-modified reduced recombinant human endostatin with cation exchange chromatography (the medium is sulfopropyl sephadex (abbreviated as SP)). A comparison chart of the activity of polyethylene glycol-modified Endostar in inhibiting HMEC cell migration; where, B is blank, L1 is the activity of Endostar at a concentration of 10 μg/ml in inhibiting HMEC cell migration, and L2 is the activity of Endostar at a concentration of 10 μg Endostar modified by polyethylene glycol/ml inhibits the activity of HMEC cell migration, L3 is the activity of nES (FT (effluent)) inhibiting HMEC cell migration, L4 is nES (25mS/cm ) component inhibits the activity of HMEC cell migration, L5 is the activity of the nES (30mS/cm) component whose administration concentration is 2 μg/ml inhibits HMEC cell migration, and L6 is the nES (40mS/cm ) component inhibits the activity of HMEC cell migration.
图4为不同给药浓度的nES(25mS/cm)组分、恩度和聚乙二醇修饰的恩度抑制HMEC细胞迁移的活性的对比图；其中，B为空白，L1为给药浓度为10μg/ml的恩度抑制HMEC细胞迁移的活性，L2为给药浓度为10μg/ml的聚乙二醇修饰的恩度抑制HMEC细胞迁移的活性，L3为给药浓度为0.1ug/ml的nES(25mS/cm)组分抑制HMEC细胞迁移的活性，L4为给药浓度为0.2μg/ml的nES(25mS/cm)组分抑制HMEC细胞迁移的活性，L5为给药浓度为0.5μg/ml的nES(25mS/cm)组分抑制HMEC细胞迁移的活性，L6为给药浓度为1.0μg/ml的nES(25mS/cm)组分抑制HMEC细胞迁移的活性。Fig. 4 is the comparison figure of the nES (25mS/cm) component of different administration concentrations, endostar and polyethylene glycol-modified endostar inhibiting the activity of HMEC cell migration; Wherein, B is blank, and L1 is that administration concentration is Endostar 10μg/ml inhibits HMEC cell migration, L2 is the activity of polyethylene glycol-modified Endostar at a concentration of 10μg/ml to inhibit HMEC cell migration, L3 is nES at a concentration of 0.1ug/ml (25mS/cm) component inhibits the activity of HMEC cell migration, L4 is the activity of nES (25mS/cm) component with an administration concentration of 0.2 μg/ml to inhibit HMEC cell migration, L5 is the administration concentration of 0.5 μg/ml The nES (25mS/cm) component of L6 inhibits the activity of HMEC cell migration, and the nES (25mS/cm) component with a concentration of 1.0 μg/ml inhibits the activity of HMEC cell migration.
图5为空白对照或聚乙二醇修饰的还原态重组人血管内皮抑制素抑制HMEC细胞增殖的活性的对比图；其中，a图为空白对照抑制HMEC细胞增殖的活性，b图为聚乙二醇修饰的还原态重组人血管内皮抑制素抑 制HMEC细胞增殖的活性。Figure 5 is a comparison chart of the activity of blank control or polyethylene glycol-modified reduced recombinant human vascular endostatin inhibiting the proliferation of HMEC cells; wherein, Figure a is the activity of the blank control to inhibit the proliferation of HMEC cells, and Figure b is the activity of polyethylene glycol-modified recombinant human vascular endostatin Alcohol-modified reduced recombinant human endostatin inhibits the proliferation of HMEC cells.
图6为现有的氧化态人血管内皮抑制素的空间结构示意图；Figure 6 is a schematic diagram of the spatial structure of the existing oxidized human vascular endostatin;
图7为现有的氧化态人血管内皮抑制素的二硫键配对情况示意图。Fig. 7 is a schematic diagram of disulfide bond pairing of existing oxidized human endostatin.
具体实施方式Detailed ways
需要说明的是，除非另外定义，本说明书使用的技术术语为所属领域的技术人员所理解的通常意义。It should be noted that, unless otherwise defined, the technical terms used in this specification have the usual meanings understood by those skilled in the art.
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药物试剂材料等，如无特殊说明，均为市售产品。The experimental methods in the following examples are conventional methods unless otherwise specified. The pharmaceutical reagent materials etc. used in the following examples, unless otherwise specified, are commercially available products.
定义definition
在本文中，新生血管形成是指新的毛细血管在原有的血管上的生成。已知多种疾病与新生血管形成有关，如肿瘤和黄斑变性。以肿瘤为例，肿瘤的生长和迁移依赖于新生血管的生成。将肿瘤中的微血管内皮细胞作为癌症治疗的靶点为治疗肿瘤提供了一种治疗方式。In this context, neovascularization refers to the generation of new capillaries on top of existing blood vessels. Numerous diseases are known to be associated with neovascularization, such as tumors and macular degeneration. Taking tumors as an example, the growth and migration of tumors depend on the formation of new blood vessels. Targeting microvascular endothelial cells in tumors as a target for cancer therapy offers a therapeutic modality for treating tumors.
在本文中，HMEC是指人微血管内皮细胞(Human Microvascular Endothelial Cell line)；HUVEC是指人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells)。In this article, HMEC refers to Human Microvascular Endothelial Cell line; HUVEC refers to Human Umbilical Vein Endothelial Cells.
在本文中，蛋白复性是指在适当条件下变性蛋白质可恢复其天然构象和生物活性，这种现象称为蛋白质的复性(renaturation)。一般复性会使因空间结构改变失活的蛋白恢复原有空间结构重新获得活性，但一种蛋白不一定只有一种空间结构。而对于血管内皮抑制素，发明人发现，通过所谓“复性”虽然使生理pH值条件下不可溶的血管内皮抑制素变得可溶，但同时也使其失去了活性，这是因为血管内皮抑制素的高活性状态反而是不可溶状态。In this paper, protein renaturation means that denatured protein can restore its natural conformation and biological activity under appropriate conditions, and this phenomenon is called protein renaturation. Generally, renaturation will restore the original spatial structure of the inactivated protein due to the change of the spatial structure and regain activity, but a protein does not necessarily have only one spatial structure. As for endostatin, the inventors found that although endostatin, which is insoluble under the condition of physiological pH value, becomes soluble through so-called "refolding", it also loses its activity at the same time, because the vascular endothelial The highly active state of inhibin is insoluble instead.
在本文中，恩度是一种已开发的以人血管内皮抑制素为活性成分的抗肿瘤药物。恩度
Figure PCTCN2022110868-appb-000001
(重组人血管内皮抑制素注射液)，是我国科学家在天然人血管内皮抑制素的基础上外加了9个氨基酸而研究开发出的1.1类抗肿瘤血管靶向药物。 In this paper, Endostar is a developed antineoplastic drug with human endostatin as the active ingredient. Endu
Figure PCTCN2022110868-appb-000001
(Recombinant human endostatin injection) is a class 1.1 anti-tumor blood vessel targeting drug researched and developed by Chinese scientists on the basis of natural human endostatin with 9 amino acids added.
本领域技术人员已知，人血管内皮抑制素分子包含四个半胱氨酸残基，这四个半胱酸残基之间可能会形成二硫键。在本文中，“还原态人血管内皮抑制素”是指包含至多一对(1对或0对)二硫键的人血管内皮抑制素。 以图7为例，在“还原态人血管内皮抑制素”中，人血管内皮抑制素分子中的Cys33与Cys173残基上的巯基没有形成二硫键，或者Cys135与Cys165残基上的巯基没有形成二硫键，或者这两个二硫键都没有形成。“氧化态人血管内皮抑制素”是指包含两对二硫键的人血管内皮抑制素。同样以图7为例，“氧化态人血管内皮抑制素”中，人血管内皮抑制素分子的Cys33与Cys173残基上的巯基形成二硫键，Cys135与Cys165残基上的巯基也形成二硫键。Those skilled in the art know that the molecule of human endostatin contains four cysteine residues, and disulfide bonds may be formed between these four cysteine residues. Herein, "reduced human endostatin" refers to human endostatin containing at most one pair (1 pair or 0 pair) of disulfide bonds. Taking Figure 7 as an example, in "reduced human endostatin", there is no disulfide bond between Cys33 and Cys173 residues in the human endostatin molecule, or there is no disulfide bond between Cys135 and Cys165 residues. A disulfide bond is formed, or neither disulfide bond is formed. "Oxidized human endostatin" refers to human endostatin comprising two pairs of disulfide bonds. Also take Figure 7 as an example, in "oxidized human endostatin", Cys33 of the human endostatin molecule forms a disulfide bond with the sulfhydryl group on the Cys173 residue, and the thiol group on the Cys135 and Cys165 residue also forms a disulfide bond. key.
在本文中，“还原态人血管内皮抑制素类似物”包括天然血管内皮抑制素的半胱氨酸突变、天然血管内皮抑制素的部分肽段、改变天然血管内皮抑制素的氨基酸序列，或者以上三种情况的组合，只要满足“包含至多一对二硫键”这一条件即可，但仍然保留还原态人血管内皮抑制素的抗血管生成和抗肿瘤活性。天然血管内皮抑制素的半胱氨酸突变是指在天然血管内皮抑制素的氨基酸序列基础上一个或多个半胱氨酸的缺失或置换；改变天然血管内皮抑制素的氨基酸序列是指在天然血管内皮抑制素的氨基酸序列基础上插入、删除或替换一部分氨基酸序列。所述还原态人血管内皮抑制素类似物包含至多一对二硫键。In this paper, "reduced human endostatin analogs" include cysteine mutations of natural endostatin, partial peptides of natural endostatin, changes in the amino acid sequence of natural endostatin, or the above The combination of the three conditions, as long as the condition of "containing at most one pair of disulfide bonds" is satisfied, but still retains the anti-angiogenesis and anti-tumor activities of the reduced human vascular endostatin. The cysteine mutation of natural vascular endostatin refers to the deletion or substitution of one or more cysteines on the basis of the amino acid sequence of natural vascular endostatin; changing the amino acid sequence of natural vascular endostatin refers to the Based on the amino acid sequence of endostatin, a part of the amino acid sequence is inserted, deleted or substituted. The reduced human endostatin analog contains at most one pair of disulfide bonds.
在本文中，“生理pH值条件下不可溶”是指还原态人血管内皮抑制素在pH7.4左右不可溶于水，例如在pH7.35～7.45之间不可溶于水。本发明的还原态人血管内皮抑制素在生理pH值条件下不可溶于水，但在其他pH值条件下是可溶于水的，例如pH小于5.5是可溶于水的。In this context, "insoluble at physiological pH" means that reduced human endostatin is insoluble in water at about pH 7.4, for example, insoluble in water at pH 7.35-7.45. The reduced human endostatin of the present invention is insoluble in water under physiological pH conditions, but is soluble in water under other pH conditions, for example, it is soluble in water when the pH is less than 5.5.
本文中的“电导率”是指溶液传导电流的能力。可以通过改变溶液中的盐浓度来改变溶液的电导率，如可以通过增加洗脱缓冲液中的盐浓度来升高洗脱缓冲液的电导率。可以用于升高电导率的盐包括但不限于氯化钾(KCl)、氯化钠(NaCl)、碳酸钾、乙酸钠、硫酸钾、硫酸钠、柠檬酸盐、磷酸盐或这些盐的混合物。"Conductivity" herein refers to the ability of a solution to conduct an electric current. The conductivity of the solution can be changed by changing the salt concentration in the solution, eg, the conductivity of the elution buffer can be increased by increasing the salt concentration in the elution buffer. Salts that can be used to increase conductivity include, but are not limited to, potassium chloride (KCl), sodium chloride (NaCl), potassium carbonate, sodium acetate, potassium sulfate, sodium sulfate, citrate, phosphate, or mixtures of these salts .
1. 本发明的还原态人血管内皮抑制素或其类似物 1. Reduced human endostatin or its analogue of the present invention
本领域技术人员通常认为，天然的且具有活性的血管内皮抑制素是有两对二硫键(如图6所示)且生理pH值条件下(例如pH7.4)可溶。天然折叠结构是血管内皮抑制素发挥功能所必要的。血管内皮抑制素分子上的四个半胱氨酸残基的巯基形成了独特的分子内巢式二硫键。这两对分子 内二硫键分别对于稳定该蛋白的二、三级结构至关重要。现有技术文献(Zhou H et al.,Contributions of disulfide bonds in a nested pattern to the structure,stability,and biological functions of endostatin,Journal of Biological Chemistry,2005,280:11303-11312)记载：血管内皮抑制素是一种球状蛋白质，其具有两对巢式模式的二硫键，分别为Cys33-Cys173和Cys135-Cys165(如图7所示)；血管内皮抑制素对内皮细胞迁移和增殖的抑制活性可能与二硫键保留的结构密切相关，尤其是二硫键Cys135-Cys165。Those skilled in the art generally believe that natural and active endostatin has two pairs of disulfide bonds (as shown in FIG. 6 ) and is soluble at physiological pH (eg, pH 7.4). The native folded structure is required for endostatin to function. The sulfhydryl groups of the four cysteine residues on the endostatin molecule form a unique intramolecular nested disulfide bond. These two pairs of intramolecular disulfide bonds are critical to stabilizing the secondary and tertiary structures of the protein, respectively. Prior art literature (Zhou H et al., Contributions of disulfide bonds in a nested pattern to the structure, stability, and biological functions of endostatin, Journal of Biological Chemistry, 2005, 280:11303-11312) records: vascular endostatin It is a globular protein with two pairs of nested disulfide bonds, Cys33-Cys173 and Cys135-Cys165 (as shown in Figure 7); the inhibitory activity of endostatin on endothelial cell migration and proliferation may be related to The structures of disulfide bond retention are closely related, especially the disulfide bond Cys135-Cys165.
本发明的发明人出人意料地发现：血管内皮抑制素的高活性结构为还原态人血管内皮抑制素，其包含至多一对二硫键，且在生理pH值条件下是不可溶的。本发明同样证明了血管内皮抑制素的活性与其半胱氨酸的状态和蛋白空间结构有关，但是本发明发现，血管内皮抑制素的高活性结构为还原态人血管内皮抑制素，而不是现有技术通常认为的氧化态人血管内皮抑制素。因此，本发明的还原态人血管内皮抑制素与氧化态人血管内皮抑制素不仅在结构上不同，而且具有更高的生物活性。这种高活性的结构并不限于其分子内包含半胱氨酸残基的还原态人血管内皮抑制素，而且包括还原态人血管内皮抑制素类似物，只要满足“包含至多一对二硫键”这一条件即可。优选地，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:1、2、3或4所示。The inventors of the present invention unexpectedly found that the highly active structure of endostatin is reduced human endostatin, which contains at most one pair of disulfide bonds and is insoluble at physiological pH. The present invention also proves that the activity of vascular endostatin is related to its cysteine state and protein space structure, but the present invention finds that the highly active structure of vascular endostatin is reduced human vascular endostatin, rather than the existing The technique generally considers the oxidized state of human endostatin. Therefore, the reduced human endostatin of the present invention is not only different in structure from the oxidized human endostatin, but also has higher biological activity. This highly active structure is not limited to reduced human endostatin containing cysteine residues in its molecule, but also includes reduced human endostatin analogs, as long as the "containing at most one pair of disulfide bonds "This condition is sufficient. Preferably, the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO: 1, 2, 3 or 4.
现有技术文献(秦叔逵，杨柳青，梁军等.，腔内应用重组人血管内皮抑制素和/或顺铂治疗恶性胸腹腔积液的前瞻性、随机对照、全国多中心Ⅲ期临床研究，[J].临床肿瘤学杂志，2017，22(3):193-202)证明了恩度单药在治疗肿瘤引起的恶性胸腹腔积液中有很好的疗效，这是目前血管内皮抑制素类药物在临床研究中证明单药有明确疗效的最佳案例。临床研究结果如下：Prior art literature (Qin Shukui, Yang Liuqing, Liang Jun, etc., Prospective, randomized controlled, national multi-center phase III clinical study of intracavitary application of recombinant human endostatin and/or cisplatin in the treatment of malignant pleural and peritoneal effusions, [ J]. Journal of Clinical Oncology, 2017, 22(3):193-202) proved that endostar monotherapy has a good effect in the treatment of malignant pleural and peritoneal effusions caused by tumors. The best case in which a drug has been proven to have a clear curative effect in clinical research. The clinical research results are as follows:
Figure PCTCN2022110868-appb-000002
Figure PCTCN2022110868-appb-000002
另有现有技术文献(尚亚娟，林英.，测定腹水巯基物对良恶性腹水的鉴别价值[J].临床肝胆病杂志，1997，13(1):2)表明，恶性胸腹腔积液中含有高浓度的还原性自由巯基，还原环境使恩度部分被还原，进而获得高活性的还原状态。这个研究结果恰恰证明了，还原态血管内皮抑制素是高活性状态的，与本申请的结果相一致。Another prior art literature (Shang Yajuan, Lin Ying., Determination of ascites sulfhydryl content to the differential value of benign and malignant ascites [J]. Clinical Hepatobiliary Diseases Journal, 1997, 13 (1): 2) shows that malignant thoracic and peritoneal The solution contains a high concentration of reducing free sulfhydryl groups, and the reducing environment makes Endostar partly reduced, thereby obtaining a highly active reduced state. This research result just proves that the reduced vascular endostatin is in a highly active state, which is consistent with the results of this application.
本发明的还原态人血管内皮抑制素类似物包括以下的至少一种或其组合：The reduced human endostatin analogs of the present invention include at least one or a combination of the following:
---在天然血管内皮抑制素的氨基酸序列基础上一个或多个(是指两个或两个以上，最多为四个)半胱氨酸的缺失或置换；--- Deletion or substitution of one or more (referring to two or more, up to four) cysteines on the basis of the amino acid sequence of natural vascular endostatin;
---天然血管内皮抑制素的部分肽段；或者--- Partial peptides of natural endostatin; or
---改变天然血管内皮抑制素的氨基酸序列；---Change the amino acid sequence of natural vascular endostatin;
其中，所述还原态人血管内皮抑制素类似物包含至多一对二硫键。Wherein, the reduced human endostatin analog contains at most one pair of disulfide bonds.
本发明的还原态人血管内皮抑制素类似物分子中可能包含四个半胱氨基酸残基、三个半胱氨酸残基、两个半胱氨酸残基、一个半胱氨酸残基或0个半胱氨酸残基，但无论存在几个半胱氨酸残基，均至多形成一对二硫键。The reduced human endostatin analog molecule of the present invention may contain four cysteine residues, three cysteine residues, two cysteine residues, one cysteine residue or 0 cysteine residues, but no matter how many cysteine residues are present, at most one pair of disulfide bonds is formed.
在本发明中，“置换”是指半胱氨酸置换为其他氨基酸，置换后的血管内皮抑制素依然具有活性，且成药性更好。In the present invention, "replacement" refers to the replacement of cysteine with other amino acids, and the substituted endostatin still has activity and better druggability.
本发明对还原态人血管内皮抑制素中的没有形成二硫键的半胱氨酸的位置不作限定，而且半胱氨酸的位置根据还原态人血管内皮抑制素的氨基酸序列不同会发生变化。优选地，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:1所示，其中二硫键形成情况包括：The present invention does not limit the position of the cysteine that does not form a disulfide bond in the reduced human endostatin, and the position of the cysteine will vary according to the amino acid sequence of the reduced human endostatin. Preferably, the amino acid sequence of the reduced human vascular endostatin is as shown in SEQ ID NO: 1, wherein the formation of disulfide bonds includes:
---还原态人血管内皮抑制素分子中的Cys33与Cys173残基上的巯基没有形成二硫键；---Cys33 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys173 residue;
---还原态人血管内皮抑制素分子中的Cys135与Cys165残基上的巯基没有形成二硫键；或者--- Cys135 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys165 residue; or
---以上两个二硫键都没有形成。---The above two disulfide bonds are not formed.
2. 本发明的还原态人血管内皮抑制素或其类似物的制备方法 2. The preparation method of reduced human vascular endostatin or its analogs of the present invention
本发明的还原态人血管内皮抑制素是从大肠杆菌表达的包涵体中直接纯化出来的。本领域技术人员公知：包涵体是外源基因在原核细胞尤其 是大肠杆菌中高效表达时形成的由膜包裹的高密度、不溶性蛋白质颗粒。细胞中的生物学活性蛋白质常以可溶蛋白或分子复合物的形式存在，功能性的蛋白质总是折叠成特定的三维结构型。包涵体内的蛋白是非折叠状态的聚集体，不具有生物学活性。对于血管内皮抑制素，通常利用基因工程的方法(例如利用大肠杆菌表达系统)得到。大肠杆菌表达系统生产的重组人血管内皮抑制素没有天然折叠结构，水溶性差，易于形成沉淀。为了得到有两对二硫键且生理pH值条件下可溶于水的重组人血管内皮抑制素，通常会对沉淀形式的重组人血管内皮抑制素进行重折叠(复性)。中国专利00107569.1(发明名称：生产内皮抑制素的方法)公开了：通过修饰人血管内皮抑制素的核苷酸编码序列，生产出N末端带有附加氨基酸序列(MGGSHHHHH)的重组人血管内皮抑制素(rhEndostatin，取名为：Endostar，中文名：恩度)，并具体公开了包涵体的处理过程：将大肠杆菌表达的包涵体溶解于含有7M盐酸胍和50mM巯基乙醇的Tris缓冲液中继续放置1小时，离心取上清液。将上清液用Ni 2+柱纯化，将从Ni 2+柱上洗脱的含内皮抑制素蛋白质的洗脱产物以1:10体积比迅速稀释于含有2.5M尿素、100mM NaCl、0.1M Tris-HCl(pH8.0)、2mM还原态谷胱甘肽和0.02mM氧化态谷胱甘肽的溶液中，以使蛋白质重折叠(复性)。在复性过程中，加入例如氧化态谷胱甘肽使半胱氨酸上的自由巯基形成二硫键。复性后的重组人血管内皮抑制素有两对二硫键，且生理pH值条件(例如pH7.4)下是可溶于水的。 The reduced human endostatin of the present invention is directly purified from the inclusion body expressed by Escherichia coli. Those skilled in the art know that inclusion bodies are high-density, insoluble protein particles wrapped by membranes formed when foreign genes are highly expressed in prokaryotic cells, especially Escherichia coli. Biologically active proteins in cells often exist in the form of soluble proteins or molecular complexes, and functional proteins are always folded into a specific three-dimensional structure. Proteins in inclusion bodies are aggregates in an unfolded state and have no biological activity. As for endostatin, it is usually obtained by genetic engineering (for example, by using the expression system of Escherichia coli). Recombinant human endostatin produced by Escherichia coli expression system has no natural folding structure, poor water solubility and easy to form precipitates. In order to obtain recombinant human endostatin having two pairs of disulfide bonds and being soluble in water at physiological pH, the precipitated recombinant human endostatin is usually refolded (refolded). Chinese patent 00107569.1 (invention name: method for producing endostatin) discloses: by modifying the nucleotide coding sequence of human endostatin, recombinant human endostatin with an additional amino acid sequence (MGGSHHHHH) at the N-terminal is produced (rhEndostatin, named: Endostar, Chinese name: Endo), and specifically disclosed the treatment process of inclusion bodies: the inclusion bodies expressed by Escherichia coli were dissolved in Tris buffer containing 7M guanidine hydrochloride and 50mM mercaptoethanol and continued to place After 1 hour, centrifuge to take the supernatant. The supernatant was purified with a Ni 2+ column, and the eluted product containing endostatin protein eluted from the Ni 2+ column was rapidly diluted at a volume ratio of 1:10 in a solution containing 2.5M urea, 100mM NaCl, 0.1M Tris -HCl (pH8.0), 2mM reduced glutathione and 0.02mM oxidized glutathione solution to refold (refold) the protein. During renaturation, free sulfhydryl groups on cysteines form disulfide bonds by adding, for example, oxidized glutathione. The refolded recombinant human endostatin has two pairs of disulfide bonds, and is soluble in water under physiological pH conditions (eg, pH 7.4).
现有技术通常认为，包涵体中的血管内皮抑制素没有天然折叠结构，是不具有活性的。而本发明中的还原态人血管内皮抑制素是从大肠杆菌表达的包涵体中直接纯化出来的，其活性远高于氧化态人血管内皮抑制素。因此，本发明提供了一种制备上述还原态人血管内皮抑制素的方法，将包含编码人血管内皮抑制素的核苷酸序列的载体转化原核表达系统(例如大肠杆菌)，经发酵表达得到包涵体，溶解并纯化包涵体，得到还原态人血管内皮抑制素。It is generally believed in the prior art that the endostatin in the inclusion body has no natural folding structure and is inactive. However, the reduced human endostatin in the present invention is directly purified from the inclusion body expressed by Escherichia coli, and its activity is much higher than that of the oxidized human endostatin. Therefore, the present invention provides a method for preparing the above-mentioned reduced human endostatin. The vector containing the nucleotide sequence encoding human endostatin is transformed into a prokaryotic expression system (such as Escherichia coli), and the inclusion is obtained through fermentation and expression. The inclusion body was dissolved and purified to obtain reduced human endostatin.
大肠杆菌表达蛋白会在N端自动加上甲硫氨酸，因此，编码人血管内皮抑制素的核苷酸序列即为编码氨基酸序列如SEQ ID NO:1或SEQ ID NO:3所示的核苷酸序列。SEQ ID NO:1与SEQ ID NO:2的区别在于：SEQ ID NO:2与SEQ ID NO:1相比在N端多一个甲硫氨酸，SEQ ID  NO:3与SEQ ID NO:4的区别在于：SEQ ID NO:4与SEQ ID NO:3相比在N端多一个甲硫氨酸。而SEQ ID NO:2与SEQ ID NO:4的区别在于：SEQ ID NO:4的N末端带有附加氨基酸序列(MGGSHHHHH)，而且SEQ ID NO:4即为现有产品恩度的氨基酸序列。本发明的还原态重组人血管内皮抑制素与恩度的区别在于，本发明的还原态重组人血管内皮抑制素未经过复性，将大肠杆菌发酵表达的包涵体溶解，直接包涵体溶解液中纯化出来。The protein expressed in Escherichia coli will automatically add methionine to the N-terminus. Therefore, the nucleotide sequence encoding human endostatin is the nucleus encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. nucleotide sequence. The difference between SEQ ID NO: 1 and SEQ ID NO: 2 is that: compared with SEQ ID NO: 1, SEQ ID NO: 2 has one more methionine at the N-terminal, and SEQ ID NO: 3 and SEQ ID NO: 4 The difference is that there is one more methionine at the N-terminal of SEQ ID NO:4 compared with SEQ ID NO:3. The difference between SEQ ID NO: 2 and SEQ ID NO: 4 is that the N-terminus of SEQ ID NO: 4 has an additional amino acid sequence (MGGSHHHHH), and SEQ ID NO: 4 is the amino acid sequence of the existing product Endu. The difference between the reduced recombinant human endostatin of the present invention and Endostar is that the reduced recombinant human endostatin of the present invention has not been refolded, and the inclusion bodies expressed by E. Purify it.
氨基酸序列1(SEQ ID NO:1)：Amino acid sequence 1 (SEQ ID NO: 1):
Figure PCTCN2022110868-appb-000003
Figure PCTCN2022110868-appb-000003
氨基酸序列2(SEQ ID NO:2)：Amino acid sequence 2 (SEQ ID NO: 2):
Figure PCTCN2022110868-appb-000004
Figure PCTCN2022110868-appb-000004
氨基酸序列3(SEQ ID NO:3)：Amino acid sequence 3 (SEQ ID NO: 3):
Figure PCTCN2022110868-appb-000005
Figure PCTCN2022110868-appb-000005
氨基酸序列4(SEQ ID NO:4)：Amino acid sequence 4 (SEQ ID NO:4):
Figure PCTCN2022110868-appb-000006
Figure PCTCN2022110868-appb-000006
3. 修饰的还原态人血管内皮抑制素或其类似物 3. Modified reduced human endostatin or its analogs
如上所述，本发明的还原态人血管内皮抑制素或其类似物相较于氧化态人血管内皮抑制素具有更高的活性，但在生理pH值状态下是不可溶于水的，无法满足成药性的要求。为了改善还原态人血管内皮抑制素或其类似物的溶解性，本发明尝试利用修饰物来修饰本发明的还原态人血管内皮抑制素或其类似物。本发明利用修饰物修饰还原态人血管内皮抑制素或其类似物，在保持还原态人血管内皮抑制素或其类似物高活性结构的同时，获得了生理pH值条件下很好的溶解性。修饰同时提高了药物的分子量，降低了肾脏滤过的速度，延长了药物的半衰期。As mentioned above, the reduced human vascular endostatin or its analogues of the present invention have higher activity than the oxidized human endostatin, but they are insoluble in water at physiological pH value and cannot satisfy Druggability requirements. In order to improve the solubility of reduced human endostatin or its analogs, the present invention attempts to use modifiers to modify the reduced human endostatin or its analogs. The present invention modifies the reduced human endostatin or its analogs with modifiers, and while maintaining the highly active structure of the reduced human endostatin or its analogs, obtains good solubility under physiological pH value conditions. The modification also increases the molecular weight of the drug, reduces the rate of renal filtration, and prolongs the half-life of the drug.
因此，本发明提供了一种修饰的还原态人血管内皮抑制素或其类似物，所述修饰物选自高分子聚合物、蛋白质分子或其片段、小分子物质中的任一种或其组合。“修饰”是指修饰物(例如高分子聚合物或小分子物质)共价修饰或非共价结合还原态人血管内皮抑制素或其类似物，或修饰物(例如蛋白质分子或其片段)与还原态人血管内皮抑制素或其类似物形成融合蛋白。在本发明的优选的实施方案中，蛋白质分子或其片段包括白蛋白、免疫球蛋白、细胞因子或它们的片段等，优选为免疫球蛋白Fc片段。免疫球蛋白Fc片段修饰的还原态人血管内皮抑制素或其类似物可通过化学修饰得到，也可通过融合表达得到。当免疫球蛋白Fc片段修饰的还原态人血管内皮抑制素通过融合表达时，免疫球蛋白Fc片段修饰的还原态人血管内皮抑制素即为免疫球蛋白Fc片段与还原态人血管内皮抑制素形成的融合蛋白。Therefore, the present invention provides a modified reduced human vascular endostatin or its analogs, wherein the modification is selected from any one of high molecular polymers, protein molecules or fragments thereof, small molecular substances or a combination thereof . "Modification" refers to the covalent modification or non-covalent combination of a modified substance (such as a high molecular polymer or a small molecular substance) or a non-covalent combination of a reduced human vascular endostatin or its analogues, or a modified substance (such as a protein molecule or a fragment thereof) combined with Reduced human endostatin or its analogs form a fusion protein. In a preferred embodiment of the present invention, protein molecules or fragments thereof include albumin, immunoglobulins, cytokines or fragments thereof, preferably Fc fragments of immunoglobulins. The reduced human endostatin or its analogs modified by the immunoglobulin Fc fragment can be obtained through chemical modification or fusion expression. When the reduced human endostatin modified by the immunoglobulin Fc fragment is expressed by fusion, the reduced human endostatin modified by the immunoglobulin Fc fragment is the formation of the immunoglobulin Fc fragment and the reduced human endostatin fusion protein.
因此，本发明提供了一种修饰的还原态人血管内皮抑制素或其类似物，使其保持高活性状态的同时生理pH值条件下可溶于水，这种修饰后可溶的还原态人血管内皮抑制素或其类似物可以测定体外细胞活性，并使其进一步开发成全身给药的药物称为可能。Therefore, the present invention provides a modified reduced human vascular endostatin or its analogues, which can maintain a high activity state and be soluble in water under physiological pH value conditions. After this modification, the soluble reduced human Endostatin or its analogues can be used to measure cell viability in vitro and make it possible to further develop drugs for systemic administration.
优选地，用高分子聚合物对还原态人血管内皮抑制素或其类似物进行修饰。更优选地，用聚乙二醇(polyethylene glycol，PEG)对还原态人血管内皮抑制素或其类似物进行修饰。聚乙二醇分子具有两亲性，既可以溶解于水，又可以溶解于大多数的有机溶剂，具有无毒、无免疫原性、在水溶液中有高溶解性等性质。将蛋白质与亲水性的高分子如聚乙二醇偶联， 可增加蛋白稳定性、减少非特异性吸附和免疫原性。当偶联物达到一定分子量时，可大大降低肾脏的排除效率，是延长蛋白质药物体内半衰期的有效方法。最初的聚乙二醇修饰都是以氨基作为反应位点，主要包括蛋白的N端α-氨基和赖氨酸残基侧链上的ε-氨基。这一类反应的产物是一个蛋白分子与一个或多个聚乙二醇分子偶联。对于赖氨酸残基侧链ε-氨基上的修饰也往往因为反应位点不特异而产生修饰后的同分异构体。目前，针对蛋白N端α-氨基和赖氨酸残基侧链ε-氨基的等电点差异，又新开发出了只针对蛋白N端修饰的聚乙二醇修饰试剂，使得修饰位点一致，修饰产物组成均一。另外，半胱氨酸上的巯基也可作为特异的修饰位点。Preferably, the reduced human endostatin or its analogues are modified with a polymer. More preferably, the reduced human endostatin or its analogs are modified with polyethylene glycol (PEG). Polyethylene glycol molecules are amphiphilic and can be dissolved in both water and most organic solvents. They are non-toxic, non-immunogenic, and highly soluble in aqueous solutions. Coupling proteins with hydrophilic polymers such as polyethylene glycol can increase protein stability, reduce non-specific adsorption and immunogenicity. When the conjugate reaches a certain molecular weight, it can greatly reduce the elimination efficiency of the kidney, and is an effective method to prolong the half-life of protein drugs in vivo. The initial polyethylene glycol modification used amino groups as reactive sites, mainly including the N-terminal α-amino group of the protein and the ε-amino group on the side chain of the lysine residue. The product of this type of reaction is the coupling of a protein molecule to one or more polyethylene glycol molecules. The modification of the ε-amino group on the side chain of lysine residues often produces modified isomers because the reaction site is not specific. At present, in view of the difference in the isoelectric point of the N-terminal α-amino group of the protein and the ε-amino group of the side chain of the lysine residue, a newly developed polyethylene glycol modification reagent that only targets the N-terminal modification of the protein makes the modification site consistent , the composition of the modified product is uniform. In addition, the sulfhydryl group on cysteine can also be used as a specific modification site.
WO 2007082483A1(发明名称：一种治疗肿瘤的药物及其应用)公开了PEG修饰的血管内皮抑制素(Endostatin)，但是该文献中使用的血管内皮抑制素为复性后的血管内皮抑制素(氧化态人血管内皮抑制素)，含有两对二硫键，生理pH值状态下是可溶于水的。作此推断的依据如下：WO 2007082483A1 (invention name: a drug for treating tumors and its application) discloses PEG-modified endostatin (Endostatin), but the endostatin used in this document is the endostatin (oxidized endostatin) after refolding Human vascular endostatin), containing two pairs of disulfide bonds, is soluble in water at physiological pH. The basis for this inference is as follows:
(1)该文献中提到“此类引入半胱氨酸作为修饰位点也是有一定的局限性，因为对某些本身带有半胱氨酸的蛋白可能会造成二硫键的错配或无法复性。”由此推断，该文献中用聚乙二醇修饰的血管内皮抑制素是经过复性的。(1) It is mentioned in the literature that "this kind of introduction of cysteine as a modification site also has certain limitations, because some proteins with cysteine may cause mismatching of disulfide bonds or It cannot be refolded." It can be inferred that the endostatin modified with polyethylene glycol in this document has been refolded.
(2)该文献中提到“在本发明中，我们惊奇的发现N端修饰的重组人血管内皮抑制素其产物在体外细胞学实验中，对内皮细胞的迁移的抑制率是未修饰蛋白的2倍或以上，既蛋白的生物活性有了很大的提高，这种体外活性明显增加的现象尚未见报道。”细胞实验所用血管内皮抑制素在生理pH值条件下必须是可溶的，否则无法用来测定活性。因此，上述表述说明该文献中所用的血管内皮抑制素在生理pH值条件下是可溶状态，也就是所谓有两对二硫键的复性状态。一般蛋白修饰后活性降低是常态，因为连接的聚乙二醇会形成空间位阻效应，而该文献中活性反而增强。据此推断，该文献中血管内皮抑制素的制备方式是大肠杆菌表达包涵体后体外复性，有部分未形成二硫键或二硫键配对不完整的血管内皮抑制素，因为聚乙二醇修饰使这部分处于还原状态的血管内皮抑制素保留了下来，而不是像通常情况下形成沉淀，其活性的提高是这部分未复性的血管内皮抑制素所致。(2) It is mentioned in this document that "in the present invention, we have surprisingly found that the N-terminally modified recombinant human endostatin product has an inhibitory rate to the migration of endothelial cells in an in vitro cytology experiment that is lower than that of the unmodified protein. 2 times or more, the biological activity of the protein has been greatly improved, and this phenomenon of significantly increased in vitro activity has not been reported.” Vascular endostatin used in cell experiments must be soluble under physiological pH conditions, otherwise Cannot be used to measure activity. Therefore, the above statement shows that the endostatin used in this document is in a soluble state at physiological pH, that is, the so-called refolding state with two pairs of disulfide bonds. Generally, it is normal for the activity to decrease after protein modification, because the linked polyethylene glycol will form a steric hindrance effect, but the activity in this literature is enhanced instead. Based on this, it can be inferred that the preparation method of endostatin in this document is that the expression of inclusion bodies in Escherichia coli is renatured in vitro, and some endostatin has not formed disulfide bonds or has incomplete disulfide bond pairing, because polyethylene glycol The modification makes this part of endostatin in the reduced state remain, instead of forming a precipitate as usual, and the increase in its activity is caused by this part of unrefolded endostatin.
由上述内容可知，现有技术中未公开聚乙二醇修饰的还原态人血管内 皮抑制素。发明人将聚乙二醇修饰的还原态人血管内皮抑制素与聚乙二醇修饰的恩度(氧化态人血管内皮抑制素)进行活性对比，实验结果证明，1、2、5μg/ml的给药浓度下，聚乙二醇修饰的还原态重组人血管内皮抑制素(nES)抑制HMEC细胞迁移的抑制率分别为28％、76％和90％，给药浓度为10μg/ml的恩度抑制HMEC细胞迁移的抑制率为48％，给药浓度为10μg/ml的聚乙二醇修饰的恩度抑制HMEC细胞迁移的抑制率为54％。这表明，聚乙二醇修饰的还原态重组人血管内皮抑制素(nES)在给药浓度仅为聚乙二醇修饰的恩度的1/5时，其抑制HMEC细胞迁移的抑制率就远高于聚乙二醇修饰的恩度抑制HMEC细胞迁移的抑制率，本发明的聚乙二醇修饰的还原态重组人血管内皮抑制素(nES)的活性为聚乙二醇修饰的恩度的5-10倍(参见实施例1)。From the foregoing, it can be seen that the reduced human endostatin modified with polyethylene glycol has not been disclosed in the prior art. The inventor compared the activity of polyethylene glycol-modified reduced human endostatin with polyethylene glycol-modified Endostar (oxidized human endostatin), and the experimental results proved that 1, 2, and 5 μg/ml At the administration concentration, the inhibitory rates of polyethylene glycol-modified reduced recombinant human endostatin (nES) to inhibit the migration of HMEC cells were 28%, 76% and 90%, respectively, and the administration concentration was 10 μg/ml Endostar The inhibition rate of inhibiting HMEC cell migration was 48%, and the inhibition rate of HMEC cell migration inhibited by polyethylene glycol-modified Endostar at a concentration of 10 μg/ml was 54%. This shows that when the reduced state recombinant human endostatin (nES) modified by polyethylene glycol is only 1/5 of that of endostar modified by polyethylene glycol, its inhibitory rate of inhibiting HMEC cell migration is far Higher than the endostar modified by polyethylene glycol inhibits the inhibition rate of HMEC cell migration, the activity of the reduced state recombinant human endostatin (nES) modified by polyethylene glycol of the present invention is that of the endostar modified by polyethylene glycol. 5-10 times (see Example 1).
本发明的实施例已经证明了，经过聚乙二醇修饰的还原态人血管内皮抑制素比经过聚乙二醇修饰的氧化态人血管内皮抑制素有更高的活性，前者活性为后者的5-10倍。本发明的实施例进一步证明了：这种高活性的聚乙二醇修饰的还原态人血管内皮抑制素可以进一步纯化，经过离子交换纯化后获得的特定组分的活性会继续提高10倍左右，即此组分的活性是经过聚乙二醇修饰的氧化态人血管内皮抑制素的50-100倍(参见实施例2)。The embodiments of the present invention have proved that the reduced human endostatin modified with polyethylene glycol has higher activity than the oxidized human endostatin modified with polyethylene glycol, and the activity of the former is higher than that of the latter. 5-10 times. The examples of the present invention further prove that the highly active polyethylene glycol-modified reduced human endostatin can be further purified, and the activity of specific components obtained after ion-exchange purification will continue to increase by about 10 times. That is, the activity of this component is 50-100 times that of the oxidized human endostatin modified with polyethylene glycol (see Example 2).
4. 修饰的还原态人血管内皮抑制素或其类似物的制备方法 4. Method for preparing modified reduced human endostatin or its analogs
在本发明中，将活化的聚乙二醇与还原态人血管内皮抑制素或其类似物混合并反应得到修饰的还原态人血管内皮抑制素或其类似物。优选地，将单甲氧基聚乙二醇丙醛与还原态人血管内皮抑制素混合，并加入还原剂氰基硼氢化钠，反应；在氰基硼氢化钠的存在下，单甲氧基聚乙二醇丙醛与还原态人血管内皮抑制素的N端氨基键的伯胺发生还原氨化反应。更优选地，单甲氧基聚乙二醇丙醛与还原态人血管内皮抑制素的物质的量比为1:1，反应的pH值为4.5～5.5，反应的时间为4～6小时，反应温度为室温。In the present invention, the activated polyethylene glycol is mixed with reduced human endostatin or its analogs and reacted to obtain modified reduced human endostatin or its analogs. Preferably, monomethoxypolyethylene glycol propionaldehyde is mixed with reduced human endostatin, and the reducing agent sodium cyanoborohydride is added for reaction; in the presence of sodium cyanoborohydride, monomethoxy Reductive amination reaction occurs between polyethylene glycol propionaldehyde and the primary amine of the N-terminal amino bond of reduced human endostatin. More preferably, the ratio of monomethoxy polyethylene glycol propionaldehyde to reduced human endostatin is 1:1, the pH value of the reaction is 4.5-5.5, and the reaction time is 4-6 hours. The reaction temperature is room temperature.
在本发明的优选的实施方案中，以体外活性为指示，通过纯化进一步富集了高活性状态的还原态人血管内皮抑制素。因此，上述制备方法还包括用阳离子交换层析(介质为磺酸基丙基葡聚糖凝胶(简称SP))纯化聚乙二醇与还原态人血管内皮抑制素的偶联产物。利用阳离子交换层析(介 质为磺酸基丙基葡聚糖凝胶(简称SP))纯化后获得特定组分(例如用电导率为25mS/cm的洗脱缓冲液洗脱的组分)的活性是经过聚乙二醇修饰的氧化态人血管内皮抑制素的50-100倍。In a preferred embodiment of the present invention, the reduced human endostatin in a highly active state is further enriched by purification, taking the in vitro activity as an indication. Therefore, the above preparation method also includes purifying the coupling product of polyethylene glycol and reduced human endostatin by cation exchange chromatography (the medium is sulfopropyl sephadex (abbreviated as SP)). Utilize cation exchange chromatography (the medium is sulfopropyl sephadex (abbreviated as SP)) to obtain specific components (for example, components eluted with an elution buffer with a conductivity of 25 mS/cm) The activity is 50-100 times that of the oxidized human endostatin modified by polyethylene glycol.
5. 药物组合物 5. Pharmaceutical composition
本发明提供了一种药物组合物，其包含上述还原态人血管内皮抑制素或其类似物或上述修饰的还原态人血管内皮抑制素或其类似物，以及药学上可接受的载体。所述药物组合物包含有效量(例如0.01-99.9重量％)的还原态人血管内皮抑制素或其类似物或修饰的还原态人血管内皮抑制素或其类似物，以及药学上可接受的载体。所述载体例如，但不限于，稀释剂(如水)、赋形剂等；粘合剂，如纤维素衍生物、明胶、聚乙烯吡咯烷酮等；填充剂如淀粉等；崩裂剂如碳酸钙、碳酸氢钠；润滑剂如硬脂酸钙或硬脂酸镁等。另外，还可以在组合物中加入其他辅助剂如香味剂和甜味剂。用于口服时，可将其制备成常规的固体制剂如片剂、粉剂或胶囊等；用于注射时，可将其制备成注射液。所述药物组合物的具体剂量可根据临床实验结果及患者的病情、年龄等由医师决定。The present invention provides a pharmaceutical composition, which comprises the above-mentioned reduced human endostatin or its analogue or the above-mentioned modified reduced human endostatin or its analogue, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprises an effective amount (for example, 0.01-99.9% by weight) of reduced human endostatin or its analogs or modified reduced human endostatin or its analogs, and a pharmaceutically acceptable carrier . Said carrier is for example, but not limited to, diluents (such as water), excipients, etc.; binders, such as cellulose derivatives, gelatin, polyvinylpyrrolidone, etc.; fillers such as starch, etc.; disintegrants such as calcium carbonate, carbonic acid sodium hydrogen; lubricants such as calcium stearate or magnesium stearate, etc. In addition, other adjuvants such as flavoring and sweetening agents can also be added to the composition. When used for oral administration, it can be prepared into conventional solid preparations such as tablets, powders or capsules, etc.; when used for injection, it can be prepared as injection solution. The specific dose of the pharmaceutical composition can be determined by a doctor according to the results of clinical experiments and the patient's condition and age.
本发明的药物组合物可以制成普通制剂，也可以是缓释制剂。所述缓释制剂选自微胶囊、水凝胶、微球、微型渗透泵或脂质体等。将上述还原态人血管内皮抑制素或其类似物或上述修饰的还原态人血管内皮抑制素或其类似物制成缓释制剂可延长还原态人血管内皮抑制素或其类似物在体内的半衰期。在使用时，将上述还原态人血管内皮抑制素或其类似物或修饰的还原态人血管内皮抑制素或其类似物放入药用载体(例如水凝胶、脂质体等)中，使得还原态人血管内皮抑制素或其类似物从药用载体中缓慢释放，在体内维持一种稳定的还原态人血管内皮抑制素或其类似物浓度。The pharmaceutical composition of the present invention can be made into common preparations or sustained-release preparations. The sustained-release preparation is selected from microcapsules, hydrogels, microspheres, miniature osmotic pumps or liposomes and the like. Making the above-mentioned reduced human endostatin or its analogs or the above-mentioned modified reduced human endostatin or its analogs into sustained-release preparations can prolong the half-life of reduced human endostatin or its analogs in vivo . When in use, the above-mentioned reduced human endostatin or its analogues or modified reduced human endostatin or its analogues are put into a pharmaceutical carrier (such as hydrogel, liposome, etc.), so that The reduced human endostatin or its analogue is slowly released from the pharmaceutical carrier, and a stable concentration of the reduced human endostatin or its analogue is maintained in the body.
6. 用途 6. Purpose
本发明的实施例已经证明了还原态人血管内皮抑制素或其类似物和聚乙二醇修饰的还原态人血管内皮抑制素或其类似物具有抑制血管内皮细胞增殖和迁移的的活性。因此，本发明的还原态人血管内皮抑制素或其类似物和聚乙二醇修饰的还原态人血管内皮抑制素或其类似物、相应的药物组合物可用于制备抗肿瘤药物。优选地，所述肿瘤选自肺癌、神经内分 泌瘤、结肠癌、骨癌、肝癌、胃癌、胰腺癌、口腔癌、乳腺癌、前列腺癌、淋巴癌、食道癌、口腔癌、鼻咽癌、宫颈癌、肉瘤、肾癌、胆癌和恶性黑色素肿瘤。另外，本发明的还原态人血管内皮抑制素或其类似物和聚乙二醇修饰的还原态人血管内皮抑制素或其类似物、相应的药物组合物可治疗其他与血管生成相关的疾病，如黄斑变性等。因此，本发明的还原态人血管内皮抑制素或其类似物和聚乙二醇修饰的还原态人血管内皮抑制素或其类似物、相应的药物组合物可用于制备抗血管生成药物。The examples of the present invention have demonstrated that reduced human endostatin or its analogs and polyethylene glycol-modified reduced human endostatin or its analogs have the activity of inhibiting the proliferation and migration of vascular endothelial cells. Therefore, the reduced human endostatin or its analogue, the polyethylene glycol modified reduced human endostatin or its analogue and the corresponding pharmaceutical composition of the present invention can be used to prepare antitumor drugs. Preferably, the tumor is selected from lung cancer, neuroendocrine tumor, colon cancer, bone cancer, liver cancer, gastric cancer, pancreatic cancer, oral cancer, breast cancer, prostate cancer, lymphatic cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, cervical cancer Carcinoma, sarcoma, kidney cancer, gallbladder cancer and malignant melanoma. In addition, the reduced human endostatin or its analogs and polyethylene glycol-modified reduced human endostatin or its analogs of the present invention, and the corresponding pharmaceutical composition can treat other diseases related to angiogenesis, such as macular degeneration. Therefore, the reduced human endostatin or its analogue, polyethylene glycol-modified reduced human endostatin or its analogue and the corresponding pharmaceutical composition of the present invention can be used to prepare anti-angiogenesis drugs.
7. 修饰的蛋白或多肽 7. Modified protein or polypeptide
本发明提出了一种新观点，即占体内蛋白很大比例的不可溶蛋白(如跨膜蛋白、胶原蛋白和纤维粘连蛋白等)在局部发挥着重要的生物学功能，由于其在生理pH值条件下溶解度差，其结构和功能很难用现有技术直接研究，也很难开发成为用于全身给药的药物，通过修饰物修饰增加这类蛋白生理pH值条件下的溶解度，可以更方便的探索其结构和功能，并能提高其成药性。因此，本发明提供了一种修饰的蛋白或多肽，其中，所述蛋白或多肽在生理pH值条件下不可溶于水，修饰物选自高分子聚合物、蛋白质分子或其片段、小分子物质中的任一种或其组合。在本发明中，可利用修饰物(例如高分子聚合物或小分子物质)共价修饰或非共价结合生理pH值条件下不可溶于水的蛋白或多肽，或修饰物(例如蛋白质分子或其片段)与生理pH值条件下不可溶于水的蛋白或多肽形成融合蛋白。在本发明的优选的实施方案中，蛋白质分子或其片段包括白蛋白、免疫球蛋白、细胞因子或它们的片段等，优选为免疫球蛋白Fc片段，免疫球蛋白Fc片段修饰的蛋白或多肽(生理pH值条件下不可溶于水)可通过化学修饰得到，也可通过融合表达得到。当免疫球蛋白Fc片段修饰的蛋白或多肽通过融合表达时，免疫球蛋白Fc片段修饰的蛋白或多肽即为免疫球蛋白Fc片段与蛋白或多肽形成的融合蛋白。The present invention proposes a new point of view, that is, insoluble proteins (such as transmembrane proteins, collagen and fibronectin, etc.) which account for a large proportion of proteins in the body play important biological functions locally. Poor solubility under certain conditions, its structure and function are difficult to directly study with the existing technology, and it is also difficult to develop into a drug for systemic administration. It is more convenient to increase the solubility of this type of protein under the condition of physiological pH value through modification. Explore its structure and function, and improve its druggability. Therefore, the present invention provides a modified protein or polypeptide, wherein the protein or polypeptide is insoluble in water under physiological pH conditions, and the modification is selected from high molecular polymers, protein molecules or fragments thereof, small molecular substances any one or combination of them. In the present invention, modifiers (such as high molecular polymers or small molecular substances) can be used to covalently modify or non-covalently bind proteins or polypeptides that are insoluble in water under physiological pH conditions, or modifiers (such as protein molecules or Fragments thereof) form fusion proteins with proteins or polypeptides that are insoluble in water under physiological pH conditions. In a preferred embodiment of the present invention, protein molecules or fragments thereof include albumin, immunoglobulins, cytokines or fragments thereof, etc., preferably immunoglobulin Fc fragments, proteins or polypeptides modified by immunoglobulin Fc fragments ( Insoluble in water under the condition of physiological pH value) can be obtained by chemical modification, and can also be obtained by fusion expression. When the protein or polypeptide modified by the Fc fragment of immunoglobulin is expressed by fusion, the protein or polypeptide modified by Fc fragment of immunoglobulin is the fusion protein formed by the Fc fragment of immunoglobulin and the protein or polypeptide.
优选地，用高分子聚合物对生理pH值条件下不可溶于水 的蛋白或多肽进行修饰。更优选地，用聚乙二醇对生理pH值条件下不可溶于水 的蛋白或多肽进行修饰。现有技术中，通常利用聚乙二醇来增加药物在体内代谢的半衰期。而本发明利用聚乙二醇修饰生理pH值条件下不可溶于水 的蛋白或多肽，能够增加生理pH值条件下不可溶于水的蛋白或多肽的溶解 度，使其在生理pH值条件下变得可溶于水。本发明的生理pH值条件下不可溶于水的蛋白或多肽包括但不限于天然蛋白或多肽或其类似物。 Preferably, the protein or polypeptide that is insoluble in water under physiological pH value conditions is modified with a high molecular polymer. More preferably, the protein or polypeptide that is insoluble in water at physiological pH is modified with polyethylene glycol. In the prior art, polyethylene glycol is usually used to increase the half-life of drug metabolism in vivo. However, the present invention utilizes polyethylene glycol to modify the water - insoluble protein or polypeptide under the physiological pH value condition, which can increase the solubility of the water-insoluble protein or polypeptide under the physiological pH value condition, so that it becomes less soluble under the physiological pH value condition. Be soluble in water. The protein or polypeptide insoluble in water under the condition of physiological pH value of the present invention includes but not limited to natural protein or polypeptide or analogues thereof.
8.动物源血管内皮抑制素的用途8. Use of animal-derived vascular endostatin
由于不同动物种属血管内皮抑制素序列上极为保守，同理动物源的血管内皮抑制素与人源的血管内皮抑制素有类似功能，可用于开发类似药物。不同物种来源血管内皮抑制素同源情况如下：Since the sequence of endostatin in different animal species is extremely conserved, similarly, endostatin from animal sources has similar functions to endostatin from human sources and can be used to develop similar drugs. The homology of endostatin from different species is as follows:
Figure PCTCN2022110868-appb-000007
Figure PCTCN2022110868-appb-000007
因此，本发明提供了动物源血管内皮抑制素在制备兽药中的用途。所述兽药可用于治疗动物的癌症，例如肝癌、食道癌、胃癌、乳腺癌、淋巴癌和恶性黑色素肿瘤等；所述兽药也可用于治疗动物的与血管生成有关的疾病，例如黄斑变性。Therefore, the present invention provides the use of animal-derived vascular endostatin in the preparation of veterinary medicine. The veterinary medicine can be used to treat cancers in animals, such as liver cancer, esophageal cancer, gastric cancer, breast cancer, lymphoma, and malignant melanoma; the veterinary medicine can also be used to treat diseases related to angiogenesis in animals, such as macular degeneration.
下面结合具体的实施例对本发明提供的技术方案做进一步的描述。下述实施例仅用于对本发明进行说明，并不会对本发明的保护范围进行限制。The technical solutions provided by the present invention will be further described below in conjunction with specific embodiments. The following examples are only used to illustrate the present invention, and do not limit the protection scope of the present invention.
对比例1聚乙二醇修饰恩度Comparative example 1 polyethylene glycol modified endostar
本对比例使用的恩度为山东先声麦得津生物制药有限公司所生产，可商购获得。将恩度透析到30mM HAc-NaAc、pH4.5-5.5缓冲液中，加入等物质量的单甲氧基聚乙二醇丙醛(mPEG-ALD，20kDa)，加入终浓度20mM的还原剂氰基硼氢化钠(NaBH 3CN)，搅拌均匀，室温静置4-6小时，SDS-PAGE电泳检测。经SDS-PAGE电泳检测，确认已成功获得聚乙二醇修饰的恩度。 Endostar used in this comparative example is produced by Shandong Xiansheng Medzin Biopharmaceutical Co., Ltd. and is commercially available. Dialyze Endostar into 30mM HAc-NaAc, pH4.5-5.5 buffer solution, add monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD, 20kDa) in equivalent amount, and add reducing agent cyanide at a final concentration of 20mM Sodium borohydride (NaBH 3 CN), stirred evenly, left at room temperature for 4-6 hours, and detected by SDS-PAGE electrophoresis. It was confirmed by SDS-PAGE electrophoresis that the polyethylene glycol-modified endostar had been successfully obtained.
实施例1聚乙二醇修饰还原态重组人血管内皮抑制素Embodiment 1 polyethylene glycol modified recombinant human endostatin in reduced state
1.还原态重组人血管内皮抑制素的制备1. Preparation of reduced recombinant human endostatin
本实施例涉及的还原态重组人血管内皮抑制素包括具有“氨基酸序列2”或“氨基酸序列4”的重组人血管内皮抑制素。将含有编码“氨基酸序列1”或“氨基酸序列3”基因的DNA片段克隆至大肠杆菌表达载体pET30a，构建后的载体转化大肠杆菌，经发酵表达的目的蛋白为包涵体(大肠杆菌表达蛋白会在N端自动加上甲硫氨酸)。将大肠杆菌表达的包涵体溶解于含有7M盐酸胍或8M尿素和20mM二硫苏糖醇(DTT)的20mM Tris-HCl缓冲液中，室温继续放置3-6小时，离心取上清液，从上清液中纯化还原态重组人血管内皮抑制素。The reduced recombinant human endostatin involved in this embodiment includes recombinant human endostatin with "amino acid sequence 2" or "amino acid sequence 4". The DNA fragment containing the gene encoding "amino acid sequence 1" or "amino acid sequence 3" was cloned into Escherichia coli expression vector pET30a, the constructed vector was transformed into Escherichia coli, and the target protein expressed by fermentation was an inclusion body (the protein expressed in Escherichia coli would be in The N-terminus automatically adds methionine). The inclusion bodies expressed by Escherichia coli were dissolved in 20mM Tris-HCl buffer solution containing 7M guanidine hydrochloride or 8M urea and 20mM dithiothreitol (DTT), left at room temperature for 3-6 hours, centrifuged to get the supernatant, from Purification of reduced recombinant human endostatin from the supernatant.
2.聚乙二醇修饰还原态重组人血管内皮抑制素2. PEG-modified reduced recombinant human endostatin
将纯化后的还原态重组人血管内皮抑制素透析到30mM HAc-NaAc、pH4.5-5.5缓冲液中，加入等物质量的单甲氧基聚乙二醇丙醛(mPEG-ALD，20kDa)，加入终浓度20mM氰基硼氢化钠(NaBH 3CN)，搅拌均匀，室温静置4-6小时，SDS-PAGE电泳检测(图1)。如图1所示，相较于未修饰的还原态重组人血管内皮抑制素，聚乙二醇修饰的还原态重组人血管内皮抑制素分子量变大，其在SDS-PAGE电泳中的迁移率明显变小，这说明聚乙二醇成功修饰了还原态重组人血管内皮抑制素。 The purified reduced recombinant human endostatin was dialyzed into 30mM HAc-NaAc, pH 4.5-5.5 buffer, and an equivalent amount of monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD, 20kDa) was added , add a final concentration of 20mM sodium cyanoborohydride (NaBH 3 CN), stir evenly, let stand at room temperature for 4-6 hours, and detect by SDS-PAGE electrophoresis (Figure 1). As shown in Figure 1, compared with unmodified reduced recombinant human endostatin, the molecular weight of polyethylene glycol-modified reduced recombinant human endostatin becomes larger, and its mobility in SDS-PAGE electrophoresis is obvious becomes smaller, which shows that polyethylene glycol has successfully modified the reduced recombinant human endostatin.
实施例2 20kDa聚乙二醇与还原态重组人血管内皮抑制素N端的偶联Example 2 Coupling of 20kDa polyethylene glycol to N-terminus of recombinant human endostatin in reduced state
本实施例涉及的还原态重组人血管内皮抑制素包括具有“氨基酸序列2”的重组人血管内皮抑制素。本实施例的还原态重组人血管内皮抑制素的制备方法同实施例1。The reduced recombinant human endostatin involved in this embodiment includes recombinant human endostatin having "amino acid sequence 2". The preparation method of the reduced recombinant human vascular endostatin in this example is the same as that in Example 1.
将还原态重组人血管内皮抑制素透析到30mM HAc-NaAc、pH4.5-5.5缓冲液中，加入等物质量的单甲氧基聚乙二醇丙醛(mPEG-ALD，20kDa)，加入终浓度20mM还原剂氰基硼氢化钠(NaBH3CN)，搅拌均匀，室温静置4-6小时，SDS-PAGE电泳检测。一个聚乙二醇与一个还原态血管内皮抑制素分子偶联，并且偶联的位点是还原态血管内皮抑制素N端的α-氨基，少量还原态血管内皮抑制素会被非特异性的多位点修饰或未修饰。这时反应液可直接用于上柱纯化除去多修饰和未修饰的还原态血 管内皮抑制素。The reduced recombinant human endostatin was dialyzed into 30mM HAc-NaAc, pH4.5-5.5 buffer solution, and the equivalent amount of monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD, 20kDa) was added, and the final Concentration of 20mM reducing agent sodium cyanoborohydride (NaBH3CN), stirring evenly, standing at room temperature for 4-6 hours, SDS-PAGE electrophoresis detection. A polyethylene glycol is coupled to a reduced endostatin molecule, and the coupling site is the α-amino group at the N-terminal of the reduced endostatin, a small amount of reduced endostatin will be non-specifically multipositioned Point decorated or undecorated. At this time, the reaction solution can be directly used for column purification to remove multi-modified and unmodified reduced endostatin.
实施例3纯化聚乙二醇修饰的还原态重组人血管内皮抑制素Example 3 Purification of polyethylene glycol modified recombinant human endostatin in reduced state
将实施例1获得的聚乙二醇修饰的还原态重组人血管内皮抑制素用阳离子交换层析(介质为磺酸基丙基葡聚糖凝胶(简称SP))纯化，分别用用不同电导率的洗脱缓冲液(30mM NaAc，pH＝5.0，含有不同浓度的NaCl)进行洗脱，分别纯化得到FT(流出液)、25、30、40mS/cm组分，其中FT组分是聚乙二醇修饰的还原态重组人血管内皮抑制素直接上样流出的液体组分，25mS/cm组分是用电导率为25mS/cm的洗脱缓冲液洗脱所得的组分，30mS/cm组分是用电导率为30mS/cm的洗脱缓冲液洗脱所得的组分，40mS/cm组分是用电导率为40mS/cm的洗脱缓冲液洗脱所得的组分。The polyethylene glycol-modified reduced-state recombinant human endostatin obtained in Example 1 was purified by cation-exchange chromatography (the medium is sulfopropyl sephadex (abbreviated as SP)), and the The elution buffer (30mM NaAc, pH=5.0, containing different concentrations of NaCl) was eluted, and purified to obtain FT (effluent), 25, 30, 40mS/cm components, wherein the FT component is polyethylene Diol-modified reduced recombinant human vascular endostatin is directly sampled and flowed out. The 25mS/cm component is the component eluted with an elution buffer with a conductivity of 25mS/cm. The 30mS/cm group Fractions are fractions eluted with an elution buffer with a conductivity of 30mS/cm, and fractions with a conductivity of 40mS/cm are fractions eluted with an elution buffer with a conductivity of 40mS/cm.
试验例1测定聚乙二醇修饰的还原态重组人血管内皮抑制素对HMEC细胞迁移的抑制活性Test Example 1 Determination of the inhibitory activity of polyethylene glycol-modified reduced recombinant human endostatin on HMEC cell migration
用HMEC细胞测定对比例1和实施例1分别制备的聚乙二醇修饰的恩度和聚乙二醇修饰的还原态重组人血管内皮抑制素对HMEC细胞迁移的抑制活性。HMEC cells were used to determine the inhibitory activity of the polyethylene glycol-modified endostar and polyethylene glycol-modified reduced recombinant human endostatin prepared in Comparative Example 1 and Example 1 on HMEC cell migration.
对数生长期HMEC细胞，取24孔板，先向每孔加入细胞培养液800μl，然后每孔加入不同浓度药物200μl，终体积为1ml。另取一块24孔板，将Transwell小室放在孔上。将HMEC细胞用0.25％胰蛋白酶-EDTA消化，离心后以培养液重悬并计数，调整细胞浓度为10 6个/ml。取160μl细胞悬液加入Transwell上室，并加入不同浓度药物40μl，孵育后的Transwell小室放到加好药物的24孔板内。将24孔板放入37℃，5％CO 2培养箱中孵育4小时。除去Transwell上室，荧光法检测24孔板内的细胞迁移情况。 For HMEC cells in the logarithmic growth phase, take a 24-well plate, first add 800 μl of cell culture medium to each well, and then add 200 μl of drugs with different concentrations to each well, and the final volume is 1 ml. Take another 24-well plate and place the Transwell chamber on the well. The HMEC cells were digested with 0.25% trypsin-EDTA, centrifuged, resuspended in culture medium and counted, and the cell concentration was adjusted to 10 6 cells/ml. Take 160 μl of cell suspension and add it to the upper chamber of the Transwell, and add 40 μl of different concentrations of drugs. After incubation, the Transwell chamber is placed in a 24-well plate filled with drugs. Place the 24-well plate in a 37 °C, 5% CO2 incubator for 4 h. The upper chamber of the Transwell was removed, and the cell migration in the 24-well plate was detected by fluorescence method.
分别测定1、2、5μg/ml给药浓度条件下，聚乙二醇修饰的还原态重组人血管内皮抑制素(nES，实施例1制备)抑制HMEC细胞迁移的活性，其抑制率分别为28％、76％、90％，阳性对照10μg/ml的恩度(Endostar)抑制率为48％，10μg/ml的聚乙二醇修饰的恩度(PEG-Endostar，对比例1制备)抑制率为54％，空白对照为缓冲液(30mM HAc-NaAc、pH5.2 Buffer)，各给药组抑制率是在空白对照的基础上计算的，计算各给药组抑制率时假设空白对照组抑制率为0(图2)。由图2可知，聚乙二醇修饰的还原态重组人血管内皮抑制素(nES)在给药浓度仅为聚乙二醇修饰的恩度的1/5时，其抑制HMEC细胞迁移的抑制率就远高于聚乙二醇修饰的恩度或恩度抑制HMEC细胞迁移的抑制率。因此，聚乙二醇修饰的还原态重组人血管内皮抑制素的活性为恩度(Endostar)或聚乙二醇修饰的恩度(PEG-Endostar)的5-10倍。Under the conditions of 1, 2, and 5 μg/ml administration concentrations, the activity of the reduced recombinant human endostatin (nES, prepared in Example 1) modified by polyethylene glycol to inhibit the migration of HMEC cells was measured, and the inhibition rates were 28% respectively. %, 76%, 90%, the positive control 10 μg/ml Endostar (Endostar) inhibition rate was 48%, and the polyethylene glycol modified Endostar (PEG-Endostar, prepared in Comparative Example 1) inhibition rate of 10 μg/ml was 54%, the blank control is a buffer solution (30mM HAc-NaAc, pH5.2 Buffer), the inhibition rate of each administration group is calculated on the basis of the blank control, and the inhibition rate of the blank control group is assumed when calculating the inhibition rate of each administration group is 0 (Figure 2). As can be seen from Figure 2, when the reduced state recombinant human endostatin (nES) modified by polyethylene glycol is only 1/5 of that of endostar modified by polyethylene glycol, it inhibits the inhibition rate of HMEC cell migration It is much higher than the inhibition rate of PEG-modified Endostar or Endostar to inhibit HMEC cell migration. Therefore, the activity of polyethylene glycol-modified reduced recombinant human endostatin is 5-10 times that of Endostar or polyethylene glycol-modified Endostar (PEG-Endostar).
试验例2测定纯化的各组分对HMEC细胞迁移的抑制活性Test example 2 measures the inhibitory activity of each component of purification to HMEC cell migration
用HMEC细胞测定实施例3制备的FT(流出液)、25、30、40mS/cm组分和对比例1制备的聚乙二醇修饰的恩度对HMEC细胞迁移的抑制活性。HMEC cells were used to measure the inhibitory activity of FT (effluent), 25, 30, 40 mS/cm components prepared in Example 3 and polyethylene glycol-modified Endostar prepared in Comparative Example 1 on HMEC cell migration.
对数生长期HMEC细胞，取24孔板，先向每孔加入细胞培养液800μl，然后每孔加入不同浓度药物200μl，终体积为1ml。另取一块24孔板，将Transwell小室放在孔上。将HMEC细胞用0.25％胰蛋白酶-EDTA消化，离心后以培养液重悬并计数，调整细胞浓度为10 6个/ml。取160μl细胞悬液加入Transwell上室，并加入不同浓度药物40μl，孵育后的Transwell小室放到加好药物的24孔板内。将24孔板放入37℃，5％CO 2培养箱中孵育4小时。除去Transwell上室，荧光法检测24孔板内的细胞迁移情况。 For HMEC cells in the logarithmic growth phase, take a 24-well plate, first add 800 μl of cell culture medium to each well, and then add 200 μl of drugs with different concentrations to each well, and the final volume is 1 ml. Take another 24-well plate and place the Transwell chamber on the well. The HMEC cells were digested with 0.25% trypsin-EDTA, centrifuged, resuspended in culture medium and counted, and the cell concentration was adjusted to 10 6 cells/ml. Take 160 μl of cell suspension and add it to the upper chamber of the Transwell, and add 40 μl of different concentrations of drugs. After incubation, the Transwell chamber is placed in a 24-well plate filled with drugs. Place the 24-well plate in a 37 °C, 5% CO2 incubator for 4 h. The upper chamber of the Transwell was removed, and the cell migration in the 24-well plate was detected by fluorescence method.
测定2ug/ml给药浓度条件下其抑制HMEC细胞迁移的活性，FT(流出液)、25、30、40mS/cm组分的抑制率分别为-6％、94％、80％、15％，阳性对照10μg/ml的恩度(Endostar)抑制率为53％，10μg/ml的聚乙二醇修饰的恩度(PEG-Endostar，对比例1制备)抑制率为58％，空白对照为缓冲液(30mM HAc-NaAc、pH5.2)，各给药组抑制率是在空白对照的基础上计算的，计算各给药组抑制率时假设空白对照组抑制率为0(图3)。由图3可知，25mS/cm组分活性最高。Measure its activity of inhibiting HMEC cell migration under the condition of 2ug/ml administration concentration, the inhibition rate of FT (effluent), 25, 30, 40mS/cm component is respectively-6%, 94%, 80%, 15%, The endostar (Endostar) inhibition rate of positive control 10 μ g/ml is 53%, the Endostar (PEG-Endostar, prepared in comparative example 1) inhibition rate of 10 μ g/ml polyethylene glycol modification is 58%, and blank control is buffer solution (30mM HAc-NaAc, pH5.2), the inhibition rate of each administration group is calculated on the basis of the blank control, and the inhibition rate of the blank control group is assumed to be 0 (Fig. 3) when calculating the inhibition rate of each administration group. It can be seen from Figure 3 that the 25mS/cm component has the highest activity.
分别测定0.1、0.2、0.5、1.0μg/ml给药浓度的25mS/cm组分抑制HMEC细胞迁移的活性，其抑制率分别为35％、73％、81％、92％，阳性对照10μg/ml的恩度(Endostar)抑制率为43％，10μg/ml的聚乙二醇修饰的恩度(PEG-Endostar，对比例1制备)抑制率为41％，空白对 照为缓冲液(30mM HAc-NaAc、pH5.2Buffer)，各给药组抑制率是在空白对照的基础上计算的，计算各给药组抑制率时假设空白对照组抑制率为0(图4)。由图4可知，25mS/cm组分在给药浓度仅为聚乙二醇修饰的恩度的1/50时，其抑制HMEC细胞迁移的抑制率就远高于聚乙二醇修饰的恩度或恩度抑制HMEC细胞迁移的抑制率。因此，25mS/cm组分(聚乙二醇修饰的还原态重组人血管内皮抑制素)的活性是恩度(Endostar)或聚乙二醇修饰的恩度(PEG-Endostar)的50-100倍，其在100-200ng/ml的浓度下就有明显的活性。The 25mS/cm components at 0.1, 0.2, 0.5, and 1.0 μg/ml concentration were measured to inhibit the migration of HMEC cells, and the inhibition rates were 35%, 73%, 81%, and 92%, respectively, and the positive control was 10 μg/ml Endostar (Endostar) inhibition rate is 43%, 10 μ g/ml polyethylene glycol modified Endostar (PEG-Endostar, prepared in comparative example 1) inhibition rate is 41%, blank control is buffer solution (30mM HAc-NaAc , pH5.2Buffer), the inhibition rate of each administration group was calculated on the basis of the blank control, and the inhibition rate of the blank control group was assumed to be 0 (Fig. 4) when calculating the inhibition rate of each administration group. It can be seen from Figure 4 that when the administration concentration of the 25mS/cm component is only 1/50 of that of polyethylene glycol-modified endostar, its inhibition rate of inhibiting HMEC cell migration is much higher than that of polyethylene glycol-modified endostar Or Endostar inhibited the inhibition rate of HMEC cell migration. Therefore, the activity of the 25mS/cm component (polyethylene glycol-modified reduced recombinant human endostatin) is 50-100 times that of Endostar or PEG-Endostar , which has obvious activity at the concentration of 100-200ng/ml.
试验例3测定聚乙二醇修饰的还原态重组人血管内皮抑制素对HMEC或HUVEC细胞增殖的抑制活性Test Example 3 Determination of the inhibitory activity of polyethylene glycol-modified reduced recombinant human endostatin on HMEC or HUVEC cell proliferation
HUVEC细胞培养传代至3-5代，待细胞状态良好准备接种。用20mM PB缓冲液稀释聚乙二醇修饰的还原态重组人血管内皮抑制素，每个梯度设三个平行孔，在96孔板内每孔加入40μl。细胞密度为6000个/ml，在前面的96孔培养板中每孔加入160μl细胞悬液，于37℃，5％的CO 2培养箱中培养48-72h，MTT法测定增殖活性。 HUVEC cells were cultured and subcultured to passage 3-5, and the cells were ready to be inoculated when the cells were in good condition. The reduced recombinant human endostatin modified with polyethylene glycol was diluted with 20 mM PB buffer solution, three parallel wells were set up for each gradient, and 40 μl was added to each well of a 96-well plate. The cell density is 6000 cells/ml, add 160μl cell suspension to each well of the previous 96-well culture plate, culture at 37°C, 5% CO 2 incubator for 48-72h, and measure the proliferation activity by MTT method.
HMEC细胞系培养基加入0.5μg/ml聚乙二醇修饰的还原态重组人血管内皮抑制素，于37℃，5％的CO 2培养箱中培养24h生长情况如图5a，空白对照缓冲液(30mM HAc-NaAc、pH5.2 Buffer)的生长情况如图5b。由图5a和图5b的对比可以看出，聚乙二醇修饰的还原态重组人血管内皮抑制素能够显著抑制HMEC细胞的增殖。 Add 0.5 μg/ml polyethylene glycol-modified reduced recombinant human endostatin to the HMEC cell line culture medium, and culture it for 24 hours at 37°C in a 5% CO2 incubator. The growth situation is shown in Figure 5a, and the blank control buffer ( The growth of 30mM HAc-NaAc, pH5.2 Buffer) is shown in Figure 5b. It can be seen from the comparison of Figure 5a and Figure 5b that the reduced recombinant human endostatin modified with polyethylene glycol can significantly inhibit the proliferation of HMEC cells.

Claims (21)

  1. 一种还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素包含至多一对二硫键，所述二硫键由人血管内皮抑制素分子的两个半胱氨酸残基上的巯基形成。A reduced human endostatin or its analogues, wherein the reduced human endostatin comprises at most one pair of disulfide bonds, the disulfide bonds are composed of two cysteines of the human endostatin molecule Sulfhydryl groups on amino acid residues are formed.
  2. 根据权利要求1所述的还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素类似物包括以下的至少一种或其组合：The reduced human endostatin or its analog according to claim 1, wherein the reduced human endostatin analog comprises at least one or a combination of the following:
    ---在天然血管内皮抑制素的氨基酸序列基础上一个或多个半胱氨酸的缺失或置换；--- Deletion or substitution of one or more cysteines on the basis of the amino acid sequence of natural vascular endostatin;
    ---天然血管内皮抑制素的部分肽段；或者--- Partial peptides of natural endostatin; or
    ---改变天然血管内皮抑制素的氨基酸序列；---Change the amino acid sequence of natural vascular endostatin;
    其中，所述还原态人血管内皮抑制素类似物包含至多一对二硫键。Wherein, the reduced human endostatin analog contains at most one pair of disulfide bonds.
  3. 根据权利要求1或2所述的还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:1、2、3或4所示。The reduced human endostatin or its analog according to claim 1 or 2, wherein the amino acid sequence of the reduced human endostatin is as shown in SEQ ID NO: 1, 2, 3 or 4.
  4. 根据权利要求3所述的还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:1所示，其中二硫键形成情况包括：The reduced human endostatin or its analog according to claim 3, wherein the amino acid sequence of the reduced human endostatin is as shown in SEQ ID NO: 1, wherein the formation of disulfide bonds includes:
    ---还原态人血管内皮抑制素分子中的Cys33与Cys173残基上的巯基没有形成二硫键；---Cys33 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys173 residue;
    ---还原态人血管内皮抑制素分子中的Cys135与Cys165残基上的巯基没有形成二硫键；或者--- Cys135 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys165 residue; or
    ---以上两个二硫键都没有形成。---The above two disulfide bonds are not formed.
  5. 一种修饰的还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素包含至多一对二硫键，所述二硫键由人血管内皮抑制素分子的两个半胱氨酸残基上的巯基形成。A modified reduced human endostatin or an analog thereof, wherein the reduced human endostatin comprises at most one pair of disulfide bonds, and the disulfide bonds are formed by two of the human endostatin molecules Sulfhydryl groups on cysteine residues are formed.
  6. 根据权利要求5所述的还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素类似物包括以下的至少一种或其组合：The reduced human endostatin or its analog according to claim 5, wherein the reduced human endostatin analog comprises at least one or a combination of the following:
    ---在天然血管内皮抑制素的氨基酸序列基础上一个或多个半胱氨酸的缺失或置换；--- Deletion or substitution of one or more cysteines on the basis of the amino acid sequence of natural vascular endostatin;
    ---天然血管内皮抑制素的部分肽段；或者--- Partial peptides of natural endostatin; or
    ---改变天然血管内皮抑制素的氨基酸序列；---Change the amino acid sequence of natural vascular endostatin;
    其中，所述还原态人血管内皮抑制素类似物包含至多一对二硫键。Wherein, the reduced human endostatin analog contains at most one pair of disulfide bonds.
  7. 根据权利要求5或6所述的修饰的还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:1、2、3或4所示。The modified reduced human endostatin or its analogue according to claim 5 or 6, wherein the amino acid sequence of the reduced human endostatin is as shown in SEQ ID NO: 1, 2, 3 or 4 Show.
  8. 根据权利要求7所述的修饰的还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:2或4所示。The modified reduced human endostatin or its analog according to claim 7, wherein the amino acid sequence of the reduced human endostatin is as shown in SEQ ID NO: 2 or 4.
  9. 根据权利要求7所述的还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:1所示，其中二硫键形成情况包括：The reduced human endostatin or its analog according to claim 7, wherein the amino acid sequence of the reduced human endostatin is as shown in SEQ ID NO: 1, wherein the formation of disulfide bonds includes:
    ---还原态人血管内皮抑制素分子中的Cys33与Cys173残基上的巯基没有形成二硫键；---Cys33 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys173 residue;
    ---还原态人血管内皮抑制素分子中的Cys135与Cys165残基上的巯基没有形成二硫键；或者--- Cys135 in the reduced human endostatin molecule does not form a disulfide bond with the sulfhydryl group on the Cys165 residue; or
    ---以上两个二硫键都没有形成。---The above two disulfide bonds are not formed.
  10. 根据权利要求5所述的修饰的还原态人血管内皮抑制素或其类似物，其中，修饰物选自高分子聚合物、蛋白质分子或其片段、小分子物质中的任一种或其组合。The modified reduced human endostatin or its analogs according to claim 5, wherein the modification is selected from any one of high molecular polymers, protein molecules or fragments thereof, small molecular substances or a combination thereof.
  11. 根据权利要求10所述的修饰的还原态人血管内皮抑制素或其类似物，其中，所述高分子聚合物为聚乙二醇。The modified reduced human endostatin or its analogue according to claim 10, wherein the high molecular polymer is polyethylene glycol.
  12. 根据权利要求11所述的修饰的还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素的氨基酸序列如SEQ ID NO:2或4所示。The modified reduced human endostatin or its analog according to claim 11, wherein the amino acid sequence of the reduced human endostatin is as shown in SEQ ID NO: 2 or 4.
  13. 根据权利要求11所述的修饰的还原态人血管内皮抑制素或其类似物，其中，一个还原态人血管内皮抑制素或其类似物分子和一个或多个聚乙二醇分子偶联。The modified reduced human endostatin or its analog according to claim 11, wherein one reduced human endostatin or its analog molecule is coupled to one or more polyethylene glycol molecules.
  14. 根据权利要求13所述的修饰的还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素或其类似物与所述聚乙二醇偶联的位点是所述还原态人血管内皮抑制素或其类似物的N端α-氨基、赖氨酸残基侧链的ε-氨基、半胱氨酸残基侧链的巯基、天冬氨酸残基侧链的 羧基、谷氨酸残基侧链的羧基中的一种或是他们的组合。The modified reduced human endostatin or its analogue according to claim 13, wherein the site where the reduced human endostatin or its analogue is coupled to the polyethylene glycol is the The N-terminal α-amino group of reduced human endostatin or its analogs, the ε-amino group of the side chain of lysine residue, the sulfhydryl group of the side chain of cysteine residue, the side chain of aspartic acid residue One of the carboxyl group of the carboxyl group of the glutamic acid residue side chain, or a combination thereof.
  15. 根据权利要求14所述的修饰的还原态人血管内皮抑制素或其类似物，其中，所述还原态人血管内皮抑制素或其类似物与所述聚乙二醇偶联的位点是所述还原态人血管内皮抑制素或其类似物N端的α-氨基。The modified reduced human endostatin or its analogue according to claim 14, wherein the site where the reduced human endostatin or its analogue is coupled to the polyethylene glycol is the The α-amino group at the N-terminal of the reduced human endostatin or its analogs.
  16. 根据权利要求11所述的修饰的还原态人血管内皮抑制素或其类似物，其中，所述聚乙二醇分子的平均分子量在1,000到100,000道尔顿之间。The modified reduced human endostatin or its analogue according to claim 11, wherein the average molecular weight of the polyethylene glycol molecule is between 1,000 and 100,000 Daltons.
  17. 根据权利要求16所述的修饰的还原态人血管内皮抑制素或其类似物，其中，所述聚乙二醇分子的平均分子量在5,000到40,000道尔顿之间。The modified reduced human endostatin or its analogue according to claim 16, wherein the polyethylene glycol molecule has an average molecular weight between 5,000 and 40,000 Daltons.
  18. 一种药物组合物，其包含权利要求1-4任一项所述的还原态人血管内皮抑制素或其类似物或权利要求5-17中任一项所述的修饰的还原态人血管内皮抑制素或其类似物，以及药学上可接受的载体。A pharmaceutical composition comprising the reduced human vascular endostatin or its analog according to any one of claims 1-4 or the modified reduced human vascular endothelium described in any one of claims 5-17 Inhibin or its analogue, and a pharmaceutically acceptable carrier.
  19. 根据权利要求18所述的药物组合物，其中，所述药物组合物为缓释制剂。The pharmaceutical composition according to claim 18, wherein the pharmaceutical composition is a sustained release preparation.
  20. 一种修饰的蛋白或多肽，其中，所述蛋白或多肽在生理pH值条件下不可溶于水，修饰后其在生理pH值条件下可溶于水；修饰物选自高分子聚合物、蛋白质分子或其片段、小分子物质中的任一种或其组合。A modified protein or polypeptide, wherein the protein or polypeptide is insoluble in water under physiological pH conditions, and after modification, it is soluble in water under physiological pH conditions; the modification is selected from high molecular polymers, protein Any one or combination of molecules or their fragments, small molecular substances.
  21. 动物源血管内皮抑制素在制备兽药中的用途。Use of animal-derived vascular endostatin in the preparation of veterinary medicine.
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ZHOU HAO, WANG WEI, LUO YONGZHANG: "Contributions of Disulfide Bonds in a Nested Pattern to the Structure, Stability, and Biological Functions of Endostatin", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 280, no. 12, 1 March 2005 (2005-03-01), US , pages 11303 - 11312, XP093034437, ISSN: 0021-9258, DOI: 10.1074/jbc.M412072200 *

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