WO2021120937A1 - 转甲状腺素蛋白进入眼内以及在制备滴剂中的应用 - Google Patents

转甲状腺素蛋白进入眼内以及在制备滴剂中的应用 Download PDF

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WO2021120937A1
WO2021120937A1 PCT/CN2020/128588 CN2020128588W WO2021120937A1 WO 2021120937 A1 WO2021120937 A1 WO 2021120937A1 CN 2020128588 W CN2020128588 W CN 2020128588W WO 2021120937 A1 WO2021120937 A1 WO 2021120937A1
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protein
transthyretin
drops
amino acid
ttr
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PCT/CN2020/128588
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English (en)
French (fr)
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辛瑜
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童妍(上海)医疗器械有限公司
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Priority claimed from CN201911302937.3A external-priority patent/CN110960687A/zh
Priority claimed from CN202010974997.6A external-priority patent/CN112043820B/zh
Priority claimed from CN202010976582.2A external-priority patent/CN111920940B/zh
Application filed by 童妍(上海)医疗器械有限公司 filed Critical 童妍(上海)医疗器械有限公司
Priority to EP20903323.2A priority Critical patent/EP3988122A4/en
Publication of WO2021120937A1 publication Critical patent/WO2021120937A1/zh
Priority to US17/579,510 priority patent/US20220168434A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61K38/38—Albumins
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic 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
    • C07K14/76—Albumins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the technical field of medicine, in particular to the application of transthyretin as a carrier for protein and/or polypeptide drugs to enter the eye through the ocular barrier.
  • the present invention also relates to transthyretin and/or transthyretin The application of a fusion protein composed of protein and protein and/or polypeptide drugs in the preparation of drops.
  • the present invention further relates to an ophthalmic preparation containing transthyretin and a preparation method thereof, as well as treatment and ocular angiogenesis and / Or the application in related ocular diseases such as ocular retinal leakage.
  • Diabetic retinopathy (Diabetic Retinopathy, DR) is referred to as "sugar net", which is a clinical manifestation of diabetic microangiopathy and one of the most serious complications of diabetes. It has now become one of the major blinding eye diseases. Mainly in the microenvironment of long-term high glucose and hypoxia in the eye, clinical symptoms such as microvascular obstruction, microangioma, hemorrhage, venous dilatation, macular edema, neovascularization, massive vitreous hemorrhage, intraocular fibrosis, and retinal detachment gradually occur.
  • Age-related macular degeneration is an aging change in the structure of the macular area.
  • the main manifestation is that the ability of retinal pigment epithelial cells to phagocytose and digest the outer segmental membrane of the optic cell decreases.
  • the residual disc membranes that have not been completely digested are retained in the basal cell protoplasm, discharged outside the cell, and deposited in Bruch Membrane, forming drusen. Due to the particularity of the structure and function of the macula, this change is more obvious. Drusen is also seen in the elderly with normal vision, but after various pathological changes secondary to this, it leads to macular degeneration.
  • choroidal capillaries enter under RPE (retinal pigment epithelium, retinal pigment epithelium) and retinal neuroepithelium through the broken Bruch membrane to form choroidal neovascularization, which is subretinal neovascularization. Due to the abnormal structure of the neovascular wall, the leakage and hemorrhage of the blood vessel result in a series of secondary pathological changes. Age-related macular degeneration mostly occurs over the age of 45, and its prevalence increases with age. It is currently an important disease that causes blindness in the elderly.
  • ROP Retinopathy of prematurity
  • the unvascularized retina develops fibrovascular tumor proliferation and contraction, and further causes stretch Sexual retinal detachment and blindness. It used to be called Terry syndrome or posterior lens fibrosis, but the latter only reflects the late manifestations of the disease.
  • the incidence of shorter pregnancy or lower birth weight can reach 60% to 80%.
  • the cause is that premature babies take excessive oxygen in the high-oxygen environment in the incubator after birth, and are in a relatively hypoxic state after being removed from the incubator.
  • the incompletely vascularized retina produces vasoconstriction and vascular proliferation to oxygen.
  • the eye barrier mainly includes the tear barrier, the cornea/conjunctival barrier, and the blood-eye barrier; while protecting the eye, multiple barriers can effectively block the intrusion of foreign chemical and biological molecules, but also provide drugs
  • the delivery has brought greater obstacles.
  • the commonly used methods of ocular administration mainly include conventional eye drops, subconjunctival injection, scleral administration, and intravitreal injection.
  • the contact time of conventional eye drops with the ocular surface is relatively short. Due to its own structure and characteristics, it is difficult for some exogenous drugs and proteins to enter the eye effectively; and subconjunctival injection, scleral administration and intravitreal injection usually bring about A certain degree of trauma. Retinal laser treatment, vitrectomy combined with silicone oil filling, these methods are more damaged, and the vision recovery is not ideal.
  • Intraocular injection will cause certain trauma, and the delivery efficiency of functional small peptides is low and the half-life is short. For this reason, it is necessary to find a safer, more stable, effective and effective eye No harm or less harmful drugs and methods of administration.
  • the present invention does not have a safe, stable, effective and non-harmful or less harmful drug delivery method and drug to the eye to treat diabetic retinopathy.
  • DR diabetic retinopathy
  • AMD age-related macular degeneration
  • ROP retinopathy of prematurity
  • TTR transthyretin
  • transthyretin can effectively cross the corneal barrier and enter the vitreous and fundus through the use of drops for eye drops instead of conventional injections in the art, and it can also effectively transport exogenous proteins.
  • suitable protein and/or peptide drugs can be selected according to the pathological characteristics of eye diseases, and delivered to the eye through transthyretin, so as to achieve the effect of treating eye diseases.
  • the field has important application prospects as an alternative to injectable drugs.
  • transthyretin can significantly inhibit ocular retinal leakage, significantly reduce the number of retinal neovascularization, and effectively relieve DR, AMD, Eye diseases such as ROP. That is, without fusion with protein and/or peptide drugs, transthyretin itself can treat or alleviate DR, AMD, ROP and other ocular diseases, and can achieve sufficient therapeutic concentration and treatment time (long half-life).
  • hyaluronic acid has a moisturizing effect, but do not know that it can increase the permeation of transthyretin.
  • the present inventors have further discovered that hyaluronic acid is used when preparing drops such as eye drops. Acid can increase the amount of transthyretin in the vitreous cavity and the fundus of the eye, thereby effectively increasing the concentration of transthyretin.
  • the first aspect of the present invention provides the application of transthyretin as a carrier for protein and/or polypeptide drugs to enter the eye through the ocular barrier.
  • the transthyretin protein is such as (a), As shown in (b) or (c):
  • (c) A protein represented by a sequence in which the amino acid sequence in (a) or (b) has been hydrophilically modified or hydrophobically modified.
  • the protein derived from (a) has 22 amino acid substitutions in the amino acid sequence in (a).
  • the protein derived from (a) has 25 amino acid substitutions in the amino acid sequence in (a).
  • the protein derived from (a) is T3, T5, I26, N27, H31, R34, A36, A37, D38, D39 of the amino acid sequence in (a) , T40, S50, E61, E63, V65, I68, K70, I73, A81, H90, E92, P102, R104, T123, K126 and/or E127 are substituted.
  • the protein derived from (a) is the amino acid sequence in (a) after T3G, T5A, I26V, N27D, H31K, R34K, A36T, D39G, T40S, Replacement of S50A, E61D, E63K, V65T, I68V, K70R, I73L, H90Y, P102H, R104H, T123S, K126Q and E127N.
  • the protein derived from (a) is the amino acid sequence in (a) after T3G, T5A, I26V, N27D, H31K, R34K, A36T, A37S, D38E, Replacement of D39G, T40S, S50A, E61D, E63K, I68V, K70R, I73L, A81T, H90F, E92D, P102H, R104H, T123S, K126Q and E127N.
  • the protein derived from (a) has 5 amino acids deleted from the amino acid sequence in (a). More preferably, in the (b), the protein derived from (a) is deleted at positions 123-127 of the amino acid sequence in (a).
  • the amino acid sequence of the protein derived from (a) is preferably as shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
  • the hydrophilic modification or hydrophobic modification is performed on the cysteine at position 10 in the amino acid sequence in (a).
  • the hydrophobic modification is modified with a long-chain hydrophobic fragment such as n-dodecane.
  • the protein is shown in the sequence in which the cysteine at position 10 of the amino acid sequence in (a) is linked to n-dodecane through maleimide. protein.
  • the 10th cysteine in the amino acid sequence in (a) can also be linked with a fluorescent label such as 5-aminofluorescein to track the modified protein.
  • the transthyretin is expressed in fusion with the protein and/or polypeptide drug; the fusion is preferably the fusion of the protein and/or polypeptide drug to the N of the transthyretin protein End or C end.
  • the fusion of the transthyretin and the protein and/or polypeptide drugs is expressed in microbial cells and purified.
  • the microbial cell may be Escherichia coli, and the Escherichia coli includes but is not limited to E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5 ⁇ , E. coli K12 or E. coli TOP10 .
  • the purification is preferably performed by removing endotoxin through an endotoxin adsorption column (for example, using Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher), and then removing residual bacterial cells through a 0.22 ⁇ m pore size filter membrane.
  • an endotoxin adsorption column for example, using Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher
  • the protein and/or polypeptide drugs include but are not limited to: lysozyme, albumin and/or EGFR antibodies, and the molecular weight does not exceed 45 kDa.
  • the lysozyme is preferably egg white lysozyme, and its GenBank accession number is AAL69327.1.
  • the albumin is preferably egg albumin.
  • the protein and/or polypeptide drugs include proteins for the treatment of ocular retinal leakage and/or retinal angiogenesis ocular diseases such as diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity And/or peptide drugs.
  • nucleotide sequence encoding the transthyretin protein is shown in SEQ ID NO: 2.
  • the transthyretin protein is expressed by using a recombinant expression vector, and the promoter in the backbone plasmid of the recombinant expression vector is a rhamnodon inducible promoter, preferably a rhaPBAD promoter.
  • the transthyretin protein is expressed by using a recombinant expression vector.
  • the backbone vector of the recombinant expression vector can be pET-21a or a vector with 25% or more homology to it, and the backbone vector of the recombinant expression vector is 25% or more.
  • the sequence of the vector with the above homology is preferably as shown in SEQ ID NO: 8.
  • nucleotide sequence of the recombinant plasmid expressing the transthyretin protein is shown in SEQ ID NO: 3.
  • the transthyretin protein can be expressed in microbial cells (in the form of transformants), and further can be purified; the microbial cells can be Escherichia coli, and the Escherichia coli includes but is not limited to E.coli BL21, E.coli BL21(DE3), E.coli JM109, E.coli DH5 ⁇ , E.coli K12 or E.coli TOP10.
  • the purification may be to remove endotoxin through an endotoxin adsorption column (for example, using Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher), and to remove residual bacterial cells through a 0.22 ⁇ m pore size filter.
  • the transthyretin when it is expressed, it is carried out by culturing a transformant containing the transthyretin gene until the OD 600 of the obtained bacterial cell reaches 1.5-2.0 (for example, 1.6, 1.7, 1.8 or 1.9) expression.
  • 1.5-2.0 for example, 1.6, 1.7, 1.8 or 1.9
  • an expression-inducing agent when the transthyretin protein is expressed, an expression-inducing agent is used to induce expression, and the mass volume percentage of the expression-inducing agent is 0.1-2%, such as 0.2%, 0.3%, 0.4%, 0.5% , 0.7%, 0.8%, 1.2% or 1.6%, the time for inducing expression is preferably 8-20h, such as 10h, 12h, 14h, 16h, 17h, 18h or 19h; the agent for inducing expression is preferably buckthorn Sugar or IPTG.
  • the application is the fusion protein obtained by fusion expression of the transthyretin and the protein and/or polypeptide drugs, and the fusion protein is expressed by eye drops (for example, prepared into drops
  • the medicine acts on the eyes, and the number of drops per day is 1-3 times, and the amount of each drop is 0.3-0.8nmol protein/eye.
  • the drops can be administered twice a day, 1 drop each time, for 3 months.
  • the drops can be administered once a day, 1 drop each time, for 5 days.
  • the drops can be administered twice a day, 1 drop each time, for 2 weeks.
  • the second aspect of the present invention provides the application of transthyretin and/or a fusion protein composed of transthyretin and a drug in the preparation of drops, the drug being a protein and/or polypeptide Medicine, the transthyretin protein is as shown in (a), (b) or (c):
  • (c) A protein represented by a sequence in which the amino acid sequence in (a) or (b) has been hydrophilically modified or hydrophobically modified.
  • the protein derived from (a) has 22 amino acid substitutions in the amino acid sequence in (a).
  • the protein derived from (a) has 25 amino acid substitutions in the amino acid sequence in (a).
  • the protein derived from (a) is T3, T5, I26, N27, H31, R34, A36, A37, D38, D39 of the amino acid sequence in (a) , T40, S50, E61, E63, V65, I68, K70, I73, A81, H90, E92, P102, R104, T123, K126 and/or E127 are substituted.
  • the protein derived from (a) has T3G, T5A, I26V, N27D, H31K, R34K, A36T, D39G, T40S, Replacement of S50A, E61D, E63K, V65T, I68V, K70R, I73L, H90Y, P102H, R104H, T123S, K126Q and E127N.
  • the protein derived from (a) is the amino acid sequence in (a) after T3G, T5A, I26V, N27D, H31K, R34K, A36T, A37S, D38E, Replacement of D39G, T40S, S50A, E61D, E63K, I68V, K70R, I73L, A81T, H90F, E92D, P102H, R104H, T123S, K126Q and E127N.
  • the protein derived from (a) has 5 amino acids deleted from the amino acid sequence in (a). More preferably, in the (b), the protein derived from (a) is deleted at positions 123-127 of the amino acid sequence in (a).
  • the amino acid sequence of the protein derived from (a) is preferably as shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
  • the hydrophilic modification or hydrophobic modification is performed on the cysteine at position 10 in the amino acid sequence in (a).
  • the hydrophobic modification is modified with a long-chain hydrophobic fragment such as n-dodecane.
  • the protein is shown in the sequence in which the cysteine at position 10 of the amino acid sequence in (a) is linked to n-dodecane through maleimide. protein.
  • the 10th cysteine in the amino acid sequence in (a) can also be linked with a fluorescent label such as 5-aminofluorescein to track the modified protein.
  • the content of the fusion protein is 4-30 ⁇ mol/L, preferably 10-15 ⁇ mol/L.
  • the content of the transthyretin is 4-30 ⁇ mol/L, preferably 5-30 ⁇ mol/L, more preferably 10-20 ⁇ mol/L, such as 10, 15, 20 ⁇ mol/L.
  • the drops also contain physiological saline.
  • the usage amount of the physiological saline can be used in accordance with the standards stipulated in the field.
  • the drops also contain a surfactant
  • the surfactant is, for example, Tween 80, and its content is preferably 5% (v/v).
  • the drops also contain hyaluronic acid, and the mass volume percentage of the hyaluronic acid is ⁇ 6%, preferably 1-4%, more preferably 2%.
  • the drops are eye drops.
  • the drops are drops that inhibit ocular retinal leakage and/or reduce the number of retinal neovascularization, and are preferably drops for the treatment of diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity.
  • the number of dripping of the drops per day is 1-3 times, and the amount of each dripping is preferably 0.3-0.8 nmol protein/eye.
  • the drops are administered twice a day, 1 drop each time, for 3 months; and/or, the drops are administered once a day, 1 drop each time, for 5 days And/or, the drops are administered twice a day, 1 drop each time, for 2 weeks.
  • nucleotide sequence encoding the transthyretin protein is shown in SEQ ID NO: 2.
  • the transthyretin protein is expressed by using a recombinant expression vector, and the promoter in the backbone plasmid of the recombinant expression vector is a rhamnodon inducible promoter, preferably a rhaPBAD promoter.
  • the transthyretin protein is expressed by using a recombinant expression vector.
  • the backbone vector of the recombinant expression vector is pET-21a or a vector that has 25% or more homology with it, and it has 25% and
  • the sequence of the above homologous vector is preferably shown in SEQ ID NO: 8.
  • nucleotide sequence of the recombinant plasmid expressing the transthyretin protein is shown in SEQ ID NO: 3.
  • the transthyretin protein can be expressed in microbial cells (in the form of transformants), and further can be purified; the microbial cells can be Escherichia coli, and the Escherichia coli includes but is not limited to E.coli BL21, E.coli BL21(DE3), E.coli JM109, E.coli DH5 ⁇ , E.coli K12 or E.coli TOP10.
  • the purification may be to remove endotoxin through an endotoxin adsorption column (for example, using Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher), and to remove residual bacterial cells through a 0.22 ⁇ m pore size filter.
  • the transthyretin when it is expressed, it is carried out by culturing a transformant containing the transthyretin gene until the OD 600 of the obtained bacterial cell reaches 1.5-2.0 (for example, 1.6, 1.7, 1.8 or 1.9) expression.
  • 1.5-2.0 for example, 1.6, 1.7, 1.8 or 1.9
  • an expression-inducing agent when the transthyretin protein is expressed, an expression-inducing agent is used to induce expression, and the mass volume percentage of the expression-inducing agent is 0.1-2%, such as 0.2%, 0.3%, 0.4%, 0.5% , 0.7%, 0.8%, 1.2% or 1.6%, the time for inducing expression is preferably 8-20h, such as 10h, 12h, 14h, 16h, 17h, 18h or 19h; the agent for inducing expression is preferably buckthorn Sugar or IPTG.
  • sequence of the fusion protein is shown in SEQ ID NO: 6 or SEQ ID NO: 7.
  • the transthyretin is expressed in fusion with the protein and/or polypeptide drug; the fusion is preferably the fusion of the protein and/or polypeptide drug to the N of the transthyretin protein Terminal or C terminal; the fusion of the transthyretin and the protein and/or polypeptide drugs is preferably expressed in microbial cells and purified.
  • the microbial cell may be Escherichia coli, and the Escherichia coli includes but is not limited to E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5 ⁇ , E. coli K12 or E. coli TOP10 .
  • the purification is preferably to remove endotoxins through an endotoxin adsorption column, and then to remove residual bacterial cells through a 0.22 ⁇ m pore size filter membrane.
  • the protein and/or polypeptide drugs include but are not limited to: lysozyme, albumin and/or EGFR antibody, and the molecular weight does not exceed 45kDa; the lysozyme is preferably egg white lysozyme, which is registered in GenBank The number is AAL69327.1; the albumin is preferably egg albumin.
  • the protein and/or polypeptide drugs include proteins for the treatment of ocular retinal leakage and/or retinal angiogenesis ocular diseases such as diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity And/or peptide drugs.
  • carboxymethyl cellulose or its salts such as sodium carboxymethyl cellulose, chondroitin sulfate or its salts such as chondroitin sulfate A sodium salt, dextran such as dextran 70, hyaluronic acid
  • dextran such as dextran 70
  • transthyretin and/or fusion proteins can contain and cooperate with each other to form an organic whole, which can be used to treat ocular diseases such as diabetic retinopathy related to ocular angiogenesis and/or ocular retinal leakage.
  • Age-related macular degeneration and/or retinopathy of prematurity through coordinated treatment, and finally obtained better therapeutic effect.
  • hyaluronic acid carboxymethylcellulose or its salts such as sodium carboxymethylcellulose, chondroitin sulfate or its salts such as chondroitin sulfate A sodium salt, dextran such as dextran 70, hyaluronic acid, etc.
  • the materials have moisturizing effects, and they can usually be used to prepare eye drops to effectively relieve eye dryness.
  • the aforementioned drops may further contain pharmaceutically acceptable excipients selected from the group consisting of "carboxymethyl cellulose or its salt, its solvate, and its pharmaceutically acceptable salt).
  • Solvate, or, its crystal form "chondroitin sulfate or its salt, its solvate, the solvate of its pharmaceutically acceptable salt, or its crystal form", “dextran or its salt, its Solvates, solvates of pharmaceutically acceptable salts thereof, or, crystalline forms thereof, and, “hyaluronic acid or salts thereof, solvates thereof, solvates of pharmaceutically acceptable salts thereof, Or, one or more of its crystal forms.
  • the carboxymethyl cellulose or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form has a viscosity of 800-1200 CP.
  • the chondroitin sulfate is chondroitin sulfate A.
  • the dextran is dextran 70.
  • the molecular weight of the dextran 70 is usually in the range of 64000-76000.
  • the molecular weight of the hyaluronic acid is 10,000 to 500,000.
  • the "pharmaceutically acceptable” generally refers to non-toxic, safe, and suitable for patient use.
  • the "patient” is preferably a mammal, more preferably a human.
  • the “salt” in the “or its salt” is usually a pharmaceutically acceptable salt, which usually refers to a compound prepared by the compound of the present invention and a relatively non-toxic, pharmaceutically acceptable acid or base. salt.
  • the base addition salt can be obtained by contacting the prototype of the compound with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include, but are not limited to: lithium salt, sodium salt, potassium salt, calcium salt, aluminum salt, magnesium salt, zinc salt, bismuth salt, ammonium salt, and diethanolamine salt.
  • the acid addition salt can be obtained by contacting the prototype of the compound with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent.
  • the pharmaceutically acceptable acids include inorganic acids, and the inorganic acids include, but are not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, and the like.
  • the pharmaceutically acceptable acids include organic acids, including but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid , Fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid , Tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (i.e.
  • the "salt" in the "carboxymethyl cellulose or its salt” can be a sodium salt or a calcium salt, for example, sodium carboxymethyl cellulose.
  • the "salt" in the "chondroitin sulfate or its salt” can be a sodium salt or a calcium salt, for example, chondroitin sulfate A sodium salt.
  • the "solvate” refers to a substance formed by combining the compound of the present invention with a stoichiometric or non-stoichiometric solvent.
  • the solvent molecules in the solvate can exist in an ordered or non-ordered arrangement.
  • the solvents include but are not limited to: water, methanol, ethanol and the like.
  • the "pharmaceutically acceptable salt” and “solvate” in the “pharmaceutically acceptable salt solvate” are as described above, and refer to the compound 1 of the present invention and the relatively non-toxic 2. Substances formed by combining with stoichiometric or non-stoichiometric solvents, prepared from pharmaceutically acceptable acids or bases.
  • the "solvate of a pharmaceutically acceptable salt” includes, but is not limited to, the hydrochloric acid monohydrate of the adjuvant of the present invention.
  • the "excipients”, “pharmaceutically acceptable salts”, “solvates” and “solvates of pharmaceutically acceptable salts”, as well as the following “compounds” and “glycosides” Can exist in crystalline or amorphous form.
  • crystal form means that the ions or molecules are arranged strictly and periodically in a three-dimensional space in a certain way, and have the regularity of periodic recurrence at a certain distance; due to the above-mentioned periodic arrangement, there may be multiple Crystal form, that is, polymorphism.
  • amorphous means that the ions or molecules present in a disorderly distribution state, that is, there is no periodic arrangement between the ions and molecules.
  • the concentration of the carboxymethyl cellulose or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form is 0-8 mg/ mL but not 0 (ie 0 ⁇ concentration ⁇ 8 mg/mL), preferably 2, 4, 6, or 8 mg/mL.
  • the concentration of the chondroitin sulfate or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form is 0-40 mg/mL but It is not 0 (ie, 0 ⁇ concentration ⁇ 40 mg/mL), preferably 10, 20, 30, or 40 mg/mL.
  • the concentration of the dextran or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form is 0-0.8 mg/mL but not It is 0 (ie, 0 ⁇ concentration ⁇ 0.8 mg/mL), preferably 0.2, 0.4, 0.6 or 0.8 mg/mL.
  • the content of the hyaluronic acid or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form in mass volume percentage is less than or equal to 6% , Preferably 1-4%, more preferably 2%.
  • the drops are composed of 10 ⁇ mol/L transthyretin, 6 mg/mL sodium carboxymethyl cellulose and physiological saline.
  • the drops are composed of 10 ⁇ mol/L transthyretin, 0.4 mg/mL dextran 70, and physiological saline.
  • the drops are composed of 10 ⁇ mol/L transthyretin, 20 mg/mL chondroitin sulfate A sodium salt and physiological saline.
  • the drops are composed of 10 ⁇ mol/L transthyretin, 2% hyaluronic acid and physiological saline.
  • the preparation method of the drops includes mixing the pharmaceutically acceptable excipients with the transthyretin and/or fusion protein.
  • the inventor also unexpectedly discovered that when transthyretin and/or fusion protein are co-administered to the eye with specific compounds, the compound can cooperate and act together with transthyretin and/or fusion protein. Therefore, it can effectively treat or alleviate ocular diseases related to ocular retinal angiogenesis and/or ocular retinal leakage, so that the treatment effect is better.
  • the aforementioned drops may further contain a compound, a pharmaceutically acceptable salt thereof, a glycoside, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystal form thereof; the compound is selected from One or more of diclofenac, vitamin A and luteolin.
  • Diclofenac and its sodium salt are non-steroidal anti-inflammatory drugs, which have obvious analgesic, anti-inflammatory and antipyretic effects.
  • Diclofenac sodium eye drops are used to treat uveitis, keratitis, and scleritis, and inhibit the formation of corneal neovascularization; treat inflammatory reactions after intraocular surgery, laser curtain plasty or various eye injuries; inhibit cataracts Miotic reaction during surgery; used for pain relief and anti-inflammatory after excimer laser keratotomy; treatment of vernal conjunctivitis, seasonal allergic conjunctivitis and other allergic eye diseases; prevention and treatment of postoperative inflammation of cataract and intraocular lens and cystoid macular edema; And promote the formation of filtering vesicles after glaucoma filtering.
  • Diclofenac sodium eye drops are used for ocular surface use or ocular open wound use.
  • the current marketed product is Difel with a content of 1 mg/mL.
  • the structural formulas of diclofenac and diclofenac sodium are as follows:
  • Vitamin A includes retinol (A1) and 3-dehydroretinol (A2). It is a type of fat-soluble vitamin and is stable to heat, acid and alkali. Vitamin A promotes growth and reproduction, maintains the normal secretion of bones, epithelial tissue, vision and mucosal epithelium, and other physiological functions. Vitamin A and its analogues can prevent precancerous lesions. When vitamin A is deficient, it is manifested as growth retardation and decreased dark adaptability, resulting in night blindness. Vitamin A eye drops are used to promote the growth of corneal cells and the treatment of dry eye.
  • the structural formulas of retinol (A1) and 3-dehydroretinol (A2) are as follows:
  • Luteolin It is a natural flavonoid compound that exists in a variety of plants. It has a variety of pharmacological activities, such as anti-inflammatory, anti-allergic, lowering uric acid, anti-tumor, anti-bacterial, anti-viral, etc. It is mainly used clinically to relieve cough, expectorant, anti-inflammatory, lower uric acid, treat cardiovascular diseases, and treat muscular atrophy spinal Cord sclerosis, SARS, hepatitis, etc. Luteolin, mostly in the form of glycosides, exists in a variety of plants. The structural formulas of luteolin and its glycoside form are as follows:
  • the salt is usually a pharmaceutically acceptable salt, and the details are as described above.
  • the pharmaceutically acceptable salt is a sodium salt, such as diclofenac sodium.
  • the glycoside is luteolin.
  • the vitamin A is vitamin A1 and/or vitamin A2.
  • the vitamin A can be purchased from Sinopharm, with the national drug code CATOCCHM700908, CAS: 68-26-8.
  • the content of diclofenac (or diclofenac sodium, for example, its sodium salt) is 5-20 ⁇ mol/L, for example, 10 ⁇ mol/L.
  • the content of vitamin A in the drops is 2-10 ⁇ mol/L, for example, 5 ⁇ mol/L.
  • the content of the luteolin is 2-10 ⁇ mol/L, for example, 5 ⁇ mol/L.
  • the drops are composed of 10 ⁇ mol/L transthyretin, excipients, 10 ⁇ mol/L diclofenac sodium and physiological saline, and the excipients are 6 mg/mL sodium carboxymethyl cellulose, 0.4 mg /mL dextran 70, 20mg/mL chondroitin sulfate A sodium salt, and 2% (mass volume percentage) hyaluronic acid.
  • the drops are composed of 10 ⁇ mol/L transthyretin, excipients, 5 ⁇ mol/L vitamin A, 5% (v/v) Tween 80 and physiological saline, and the excipients are 6 mg /mL sodium carboxymethylcellulose, 0.4mg/mL dextran 70, 20mg/mL chondroitin sulfate A sodium salt, and 2% (mass volume percentage) hyaluronic acid.
  • the drops are composed of 10 ⁇ mol/L transthyretin, excipients, 5 ⁇ mol/L luteolin and physiological saline, and the excipients are 6 mg/mL sodium carboxymethyl cellulose, 0.4 Any one of mg/mL dextran 70, 20 mg/mL chondroitin sulfate A sodium salt, and 2% (mass volume percentage) hyaluronic acid.
  • the preparation method of the drops includes mixing the compound with the transthyretin and/or fusion protein.
  • the third aspect of the present invention provides a drop (for example, in the form of eye drops), which contains transthyretin and/or a fusion protein composed of transthyretin and a drug;
  • the drug is a protein and/or polypeptide drug, and the transthyretin protein is as shown in (a), (b) or (c):
  • (c) A protein represented by a sequence in which the amino acid sequence in (a) or (b) has been hydrophilically modified or hydrophobically modified.
  • the protein derived from (a) has 22 amino acid substitutions in the amino acid sequence in (a).
  • the protein derived from (a) has 25 amino acid substitutions in the amino acid sequence in (a).
  • the protein derived from (a) is T3, T5, I26, N27, H31, R34, A36, A37, D38, D39 of the amino acid sequence in (a) , T40, S50, E61, E63, V65, I68, K70, I73, A81, H90, E92, P102, R104, T123, K126 and/or E127 are substituted.
  • the protein derived from (a) has T3G, T5A, I26V, N27D, H31K, R34K, A36T, D39G, T40S, Replacement of S50A, E61D, E63K, V65T, I68V, K70R, I73L, H90Y, P102H, R104H, T123S, K126Q and E127N.
  • the protein derived from (a) has T3G, T5A, I26V, N27D, H31K, R34K, A36T, A37S, D38E, Replacement of D39G, T40S, S50A, E61D, E63K, I68V, K70R, I73L, A81T, H90F, E92D, P102H, R104H, T123S, K126Q and E127N.
  • the protein derived from (a) has 5 amino acids deleted from the amino acid sequence in (a). More preferably, in the (b), the protein derived from (a) is deleted at positions 123-127 of the amino acid sequence in (a).
  • the amino acid sequence of the protein derived from (a) is preferably as shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
  • the hydrophilic modification or hydrophobic modification is performed on the cysteine at position 10 in the amino acid sequence in (a).
  • the hydrophobic modification is modified with a long-chain hydrophobic fragment such as n-dodecane.
  • the protein is shown in the sequence in which the cysteine at position 10 of the amino acid sequence in (a) is linked to n-dodecane through maleimide. protein.
  • the 10th cysteine in the amino acid sequence in (a) can also be linked with a fluorescent label such as 5-aminofluorescein to track the modified protein.
  • the content of the fusion protein is 4-30 ⁇ mol/L, preferably 10-15 ⁇ mol/L.
  • the content of transthyretin is 4-30 ⁇ mol/L, preferably 5-30 ⁇ mol/L, more preferably 10-20 ⁇ mol/L, such as 10, 15 , 20 ⁇ mol/L.
  • the drops also contain physiological saline.
  • the usage amount of the physiological saline can be used in accordance with the standards stipulated in the field.
  • the drops also contain a surfactant
  • the surfactant is, for example, Tween 80, and its content is preferably 5% (v/v).
  • the drops also contain hyaluronic acid, and the mass volume percentage of the hyaluronic acid is ⁇ 6%, preferably 1-4%, more preferably 2%.
  • the drops (for example, in the form of eye drops) further contain physiological saline and 1-6% hyaluronic acid.
  • the drops (for example, in the form of eye drops) further contain physiological saline and 1-4% hyaluronic acid.
  • the drops (for example, in the form of eye drops) further contain physiological saline and 2% hyaluronic acid.
  • the drops are eye drops.
  • the drops are drops that inhibit ocular retinal leakage and/or reduce the number of retinal neovascularization, and are preferably drops for the treatment of diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity.
  • the number of dripping of the drops per day is 1-3 times, and the amount of each dripping is preferably 0.3-0.8 nmol protein/eye.
  • the drops are administered twice a day, 1 drop each time, for 3 months; and/or, the drops are administered once a day, 1 drop each time, for 5 days And/or, the drops are administered twice a day, 1 drop each time, for 2 weeks.
  • nucleotide sequence encoding the transthyretin protein is shown in SEQ ID NO: 2.
  • the transthyretin protein is expressed by using a recombinant expression vector, and the promoter in the backbone plasmid of the recombinant expression vector is a rhamnodon inducible promoter, preferably a rhaPBAD promoter.
  • the transthyretin protein is expressed by using a recombinant expression vector.
  • the backbone vector of the recombinant expression vector is pET-21a or a vector that has 25% or more homology with it, and it has 25% and
  • the sequence of the above homologous vector is preferably shown in SEQ ID NO: 8.
  • nucleotide sequence of the recombinant plasmid expressing the transthyretin protein is shown in SEQ ID NO: 3.
  • the transthyretin protein can be expressed in microbial cells (in the form of transformants), and further can be purified; the microbial cells can be Escherichia coli, and the Escherichia coli includes but is not limited to E.coli BL21, E.coli BL21(DE3), E.coli JM109, E.coli DH5 ⁇ , E.coli K12 or E.coli TOP10.
  • the purification may be to remove endotoxin through an endotoxin adsorption column (for example, using Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher), and to remove residual bacterial cells through a 0.22 ⁇ m pore size filter.
  • the transthyretin when it is expressed, it is carried out by culturing a transformant containing the transthyretin gene until the OD 600 of the obtained bacterial cell reaches 1.5-2.0 (for example, 1.6, 1.7, 1.8 or 1.9) expression.
  • 1.5-2.0 for example, 1.6, 1.7, 1.8 or 1.9
  • an expression-inducing agent when the transthyretin protein is expressed, an expression-inducing agent is used to induce expression, and the mass volume percentage of the expression-inducing agent is 0.1-2%, such as 0.2%, 0.3%, 0.4%, 0.5% , 0.7%, 0.8%, 1.2% or 1.6%, the time for inducing expression is preferably 8-20h, such as 10h, 12h, 14h, 16h, 17h, 18h or 19h; the agent for inducing expression is preferably cascara Sugar or IPTG.
  • sequence of the fusion protein is shown in SEQ ID NO: 6 or SEQ ID NO: 7.
  • the transthyretin is expressed in fusion with the protein and/or polypeptide drug; the fusion is preferably the fusion of the protein and/or polypeptide drug to the N of the transthyretin protein Terminal or C terminal; the fusion of the transthyretin and the protein and/or polypeptide drugs is preferably expressed in microbial cells and purified.
  • the microbial cell may be Escherichia coli, and the Escherichia coli includes but is not limited to E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5 ⁇ , E. coli K12 or E. coli TOP10 .
  • the purification is preferably to remove endotoxins through an endotoxin adsorption column, and then to remove residual bacterial cells through a 0.22 ⁇ m pore size filter membrane.
  • the protein and/or polypeptide drugs include but are not limited to: lysozyme, albumin and/or EGFR antibody, and the molecular weight does not exceed 45kDa; the lysozyme is preferably egg white lysozyme, which is registered in GenBank The number is AAL69327.1; the albumin is preferably egg albumin.
  • the protein and/or polypeptide drugs include proteins for the treatment of ocular retinal leakage and/or retinal angiogenesis ocular diseases such as diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity And/or peptide drugs.
  • the content of transthyretin in the above drops is 5-20 ⁇ mol/L, twice a day, 1 drop each time, for 3 months.
  • the transthyretin content in the above drops is 10-15 ⁇ mol/L, twice a day.
  • the transthyretin content in the above drops is 10 ⁇ mol/L, twice a day.
  • the transthyretin content in the above drops is 5-20 ⁇ mol/L, once a day, one drop each time, for 5 days.
  • the transthyretin content in the above drops is 10-15 ⁇ mol/L, once a day.
  • the transthyretin content in the above drops is 10 ⁇ mol/L, once a day.
  • the transthyretin content in the above drops is 5-20 ⁇ mol/L, twice a day, 1 drop each time, for 2 weeks.
  • the transthyretin content in the above drops is 10-15 ⁇ mol/L, twice a day.
  • the transthyretin content in the above drops is 10 ⁇ mol/L, twice a day.
  • the listed eye drops and the content of transthyretin in the drops are only suggestions.
  • the dosage when appropriately adjusted according to the dosage and content of the present invention for administration to a suitable subject should also fall within the protection scope of the present invention.
  • carboxymethyl cellulose or its salts such as sodium carboxymethyl cellulose, chondroitin sulfate or its salts such as chondroitin sulfate A sodium salt, dextran such as dextran 70, hyaluronic acid
  • dextran such as dextran 70
  • transthyretin and/or fusion proteins can contain and cooperate with each other to form an organic whole, which can be used to treat ocular diseases such as diabetic retinopathy related to ocular angiogenesis and/or ocular retinal leakage.
  • Age-related macular degeneration and/or retinopathy of prematurity through coordinated treatment, and finally obtained better therapeutic effect.
  • carboxymethyl cellulose or its salts such as sodium carboxymethyl cellulose
  • chondroitin sulfate or its salts such as chondroitin sulfate A sodium salt
  • dextran such as dextran 70
  • the aforementioned drops may further contain pharmaceutically acceptable excipients selected from the group consisting of "carboxymethyl cellulose or its salt, its solvate, and its pharmaceutically acceptable salt).
  • Solvate, or, its crystal form "chondroitin sulfate or its salt, its solvate, the solvate of its pharmaceutically acceptable salt, or its crystal form", “dextran or its salt, its Solvates, solvates of pharmaceutically acceptable salts thereof, or, crystal forms thereof, and “hyaluronic acid or salts thereof, solvates thereof, solvates of pharmaceutically acceptable salts thereof, or , One or more of its crystal forms.
  • the carboxymethyl cellulose or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form has a viscosity of 800-1200 CP.
  • the chondroitin sulfate is chondroitin sulfate A.
  • the dextran is dextran 70.
  • the molecular weight of the dextran 70 is usually in the range of 64000-76000.
  • the molecular weight of the hyaluronic acid is 10,000 to 500,000.
  • the "salt" in the "carboxymethyl cellulose or its salt” can be a sodium salt or a calcium salt, for example, sodium carboxymethyl cellulose.
  • the "salt" in the "chondroitin sulfate or its salt” can be a sodium salt or a calcium salt, for example, chondroitin sulfate A sodium salt.
  • the concentration of the carboxymethyl cellulose or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form is 0-8 mg/ mL but not 0 (ie 0 ⁇ concentration ⁇ 8 mg/mL), preferably 2, 4, 6, or 8 mg/mL.
  • the concentration of the chondroitin sulfate or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form is 0-40 mg/mL but It is not 0 (ie, 0 ⁇ concentration ⁇ 40 mg/mL), preferably 10, 20, 30, or 40 mg/mL.
  • the concentration of the dextran or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form is 0-0.8 mg/mL but not It is 0 (ie, 0 ⁇ concentration ⁇ 0.8 mg/mL), preferably 0.2, 0.4, 0.6 or 0.8 mg/mL.
  • the content of the hyaluronic acid or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form in mass volume percentage is less than or equal to 6% , Preferably 1-4%, more preferably 2%.
  • the drops are composed of 10 ⁇ mol/L transthyretin, 6 mg/mL sodium carboxymethyl cellulose and physiological saline.
  • the drops are composed of 10 ⁇ mol/L transthyretin, 0.4 mg/mL dextran 70, and physiological saline.
  • the drops are composed of 10 ⁇ mol/L transthyretin, 20 mg/mL chondroitin sulfate A sodium salt and physiological saline.
  • the drops are composed of 10 ⁇ mol/L transthyretin, 2% hyaluronic acid and physiological saline.
  • the preparation method of the drops includes mixing the pharmaceutically acceptable excipients with the transthyretin and/or fusion protein.
  • the inventor also unexpectedly discovered that when transthyretin and/or fusion protein are co-administered to the eye with specific compounds, the compound can cooperate and act together with transthyretin and/or fusion protein. Therefore, it can effectively treat or alleviate ocular diseases related to ocular retinal angiogenesis and/or ocular retinal leakage, so that the treatment effect is better.
  • the aforementioned drops may further contain a compound, a pharmaceutically acceptable salt thereof, a glycoside, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystal form thereof; the compound is selected from One or more of diclofenac, vitamin A and luteolin.
  • the salt is usually a pharmaceutically acceptable salt, and the details are as described above.
  • the pharmaceutically acceptable salt is a sodium salt, such as diclofenac sodium.
  • the glycoside is luteolin.
  • the vitamin A is vitamin A1 and/or vitamin A2.
  • the vitamin A can be purchased from Sinopharm, with the national drug code CATOCCHM700908, CAS: 68-26-8.
  • the content of diclofenac (or diclofenac sodium, for example, its sodium salt) is 5-20 ⁇ mol/L, for example, 10 ⁇ mol/L.
  • the content of vitamin A in the drops is 2-10 ⁇ mol/L, for example, 5 ⁇ mol/L.
  • the content of the luteolin is 2-10 ⁇ mol/L, for example, 5 ⁇ mol/L.
  • the drops are composed of 10 ⁇ mol/L transthyretin, excipients, 10 ⁇ mol/L diclofenac sodium and physiological saline, and the excipients are 6 mg/mL sodium carboxymethyl cellulose, 0.4 mg /mL dextran 70, 20mg/mL chondroitin sulfate A sodium salt, and 2% (mass volume percentage) hyaluronic acid.
  • the drops are composed of 10 ⁇ mol/L transthyretin, excipients, 5 ⁇ mol/L vitamin A, 5% (v/v) Tween 80 and physiological saline, and the excipients are 6 mg /mL sodium carboxymethylcellulose, 0.4mg/mL dextran 70, 20mg/mL chondroitin sulfate A sodium salt, and 2% (mass volume percentage) hyaluronic acid.
  • the drops are composed of 10 ⁇ mol/L transthyretin, excipients, 5 ⁇ mol/L luteolin and physiological saline, and the excipients are 6 mg/mL sodium carboxymethyl cellulose, 0.4 Any one of mg/mL dextran 70, 20 mg/mL chondroitin sulfate A sodium salt, and 2% (mass volume percentage) hyaluronic acid.
  • the preparation method of the drops includes mixing the compound with the transthyretin and/or fusion protein.
  • the transthyretin is a tetrameric carrier protein that can transport thyroid hormones in plasma and cerebrospinal fluid. Studies have found that the entry of transthyretin into cells is mediated by the high-density lipoprotein receptor SRB1 (Landers, KA, et al., Transthyretin uptake in placental cells is regulated by the high-density lipoprotein receptor, scavenger receptor class B member 1. Mol Cell Endocrinol, 2018.474: p.89-96).
  • the surface of the tetramer of transthyretin has a significant hydrophilic domain (dark) and hydrophobic domain (light), and the core hydrophobic domain of transthyretin Thyroxine molecules capable of carrying strong hydrophobicity span across various cells (see Figure 2).
  • the amino acid sequence of transthyretin from different species is highly conserved.
  • the amino acid sequence similarity between human transthyretin and transthyretin derived from SD rats and C57BL/6 mice is >95% (see Figure 3). ).
  • the inventors found that the recombinant expression level of transthyretin in Escherichia coli was extremely low, and most of them were in the form of insoluble inclusion bodies. Commonly used methods to increase protein expression include expression vector selection, fermentation conditions optimization (temperature, pH, time, inducer concentration, etc.), or molecular chaperone co-expression assistance, etc. However, the inventors tried the above-known methods and failed to effectively improve The expression level of transthyretin in Escherichia coli.
  • the present inventors found that based on the pET-21a plasmid, after replacing the promoter (such as the T7 promoter) on the plasmid with the Rhamnus tangularis inducible (such as rhaPBAD promoter), or to code the nucleotide sequence of TTR After sub-optimization (nucleotide sequence shown in SEQ ID NO: 2), or after sequence reconstruction of the pET-21a plasmid itself, or the OD value when expressing the protein, the amount of reagents that induce protein expression, and the expression induction After optimization of the time, the efficient production plasmid of the mature transthyretin fragment (NCBI Reference Sequence: NP_000362.1) can be constructed, so that the expression of the final transthyretin is significantly increased.
  • the promoter such as the T7 promoter
  • Rhamnus tangularis inducible such as rhaPBAD promoter
  • the present invention further provides a transthyretin-expressing gene, a recombinant expression vector and a transformant containing it, a recombinant plasmid for expressing transthyretin, and a method for expressing transthyretin Wait.
  • a transthyretin-expressing gene of the present invention or the recombinant plasmid expressing transthyretin is used for expression, or the transthyretin expression method of the present invention is used, the expression level of transthyretin is significantly increased . specific:
  • the fourth aspect of the present invention also provides a gene expressing transthyretin, and the nucleotide sequence of the gene is shown in SEQ ID NO: 2.
  • the fifth aspect of the present invention also provides a recombinant expression vector containing the gene as described in the fourth aspect of the present invention.
  • the promoter in the backbone vector of the recombinant expression vector is a rhamnodon inducible promoter; the rhamnodon inducible promoter is preferably a rhaPBAD promoter.
  • the backbone vector of the recombinant expression vector is pET-21a or a vector having 25% or more homology with pET-21a, and the sequence of the vector having 25% or more homology with pET-21a Preferably, it is shown in SEQ ID NO: 8.
  • nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO: 3.
  • the sixth aspect of the present invention also provides a recombinant plasmid, the sequence of which is shown in SEQ ID NO: 8.
  • the seventh aspect of the present invention also provides a recombinant plasmid expressing transthyretin, the recombinant plasmid comprising a backbone plasmid and an expression fragment of transthyretin,
  • the promoter in the backbone plasmid is a rhamnodon inducible promoter; and/or, the backbone vector of the backbone plasmid is pET-21a or a vector with 25% or more homology to it.
  • the sequence of a vector with 25% and above homology is preferably as shown in SEQ ID NO: 8.
  • nucleotide sequence encoding the expressed fragment of transthyretin is shown in SEQ ID NO: 2.
  • the buckthorn inducible promoter is rhaPBAD promoter.
  • nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO: 3.
  • the eighth aspect of the present invention also provides a transformant, which includes the gene as described in the fourth aspect of the present invention, or the recombinant expression vector as described in the fifth aspect of the present invention, or as described in the sixth or seventh aspect of the present invention.
  • the gene as described in the fourth aspect of the present invention, the recombinant expression vector as described in the fifth aspect of the present invention, or the recombinant plasmid as described in the sixth or seventh aspect of the present invention is introduced into a host, and the host is Escherichia coli, preferably E. coli BL21 (DE3), E. coli TG1, E. coli JM109, E. coli DH5 ⁇ , E. coli K12 or E. coli TOP10.
  • Escherichia coli preferably E. coli BL21 (DE3), E. coli TG1, E. coli JM109, E. coli DH5 ⁇ , E. coli K12 or E. coli TOP10.
  • the ninth aspect of the present invention also provides a method for preparing transthyretin, which includes the following steps:
  • the expression is expressed when the transformant is cultured until the OD 600 of the obtained bacterial cell reaches 1.5-2.0 (for example, 1.6, 1.7, 1.8 or 1.9).
  • the expression is induced expression by using an agent for inducing expression
  • the mass volume percentage of the agent for inducing expression is 0.1-2%, such as 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.8 %, 1.2% or 1.6%
  • the time for inducing expression is preferably 8-20h, such as 10h, 12h, 14h, 16h, 17h, 18h or 19h;
  • the agent for inducing expression is preferably rhamnose or IPTG.
  • the tenth aspect of the present invention also provides a transthyretin expression method, which includes the steps of obtaining a transformant containing a transthyretin gene and screening, expressing and purifying the transthyretin.
  • the expression is induced expression by using an agent for inducing expression
  • the mass volume percentage of the agent for inducing expression is 0.1-2%, such as 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.8 %, 1.2% or 1.6%
  • the time for inducing expression is preferably 8-20h, such as 10h, 12h, 14h, 16h, 17h, 18h or 19h;
  • the agent for inducing expression is preferably rhamnose or IPTG.
  • transthyretin can significantly inhibit ocular retinal leakage, significantly reduce the number of retinal neovascularization, and is effective Relieve DR, AMD, ROP and other eye diseases. Therefore, the present invention further provides the application of transthyretin in the preparation of drugs for inhibiting ocular retinal leakage and/or reducing the number of retinal neovascularization, for example, in the preparation and treatment of diabetic retinopathy, age-related macular degeneration and/ Or the application of drugs for eye diseases such as retinopathy of prematurity.
  • the eleventh aspect of the present invention also provides the use of transthyretin and/or the drops as described in the third aspect of the present invention in the preparation of drugs for inhibiting ocular retinal leakage and/or reducing the number of retinal neovascularization, preferably It is used in the preparation of medicines for treating age-related macular degeneration, retinopathy of prematurity and/or diabetic retinopathy.
  • the transthyretin is present in a non-injectable dosage form
  • the non-injectable dosage form is preferably a lotion or drops
  • the lotion is preferably an ointment (such as ophthalmic ointment) or a gel (such as ophthalmic gel).
  • Glue Glue
  • the drops are, for example, eye drops (the components, dosage, etc. in the drops may be as described above).
  • the gene as described in the fourth aspect of the present invention, the recombinant expression vector as described in the fifth aspect of the present invention, or the recombinant plasmid as described in the sixth or seventh aspect of the present invention is used, as described in the eighth aspect of the present invention.
  • the transformant expresses the transthyretin.
  • the twelfth aspect of the present invention also provides the application of transthyretin and/or the drops according to the third aspect of the present invention in the treatment of ocular diseases.
  • the ocular diseases are preferably ocular retinal leakage and/or ocular diseases related to retinal angiogenesis, and more preferably diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity.
  • the transthyretin is preferably as described above.
  • the conditions, dosage, administration method, etc. used in the application can all be applied as described above.
  • the thirteenth aspect of the present invention also provides a method for treating ocular retinal leakage and/or retinal angiogenesis ocular diseases such as diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity, etc., which includes administration
  • ocular retinal leakage and/or retinal angiogenesis ocular diseases such as diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity, etc.
  • the conditions, dosage, administration method, etc. treated in the method can all be performed as described above.
  • the fourteenth aspect of the present invention also provides a method for using the above-mentioned transthyretin to transport foreign proteins and/or polypeptides into the eye through the ocular barrier.
  • the method for preparing the above-mentioned fusion protein is: (1) linking transthyretin and drug protein and/or polypeptide coding gene to pET 21a(+) plasmid to obtain recombinant Plasmid; (2) Transform the recombinant plasmid constructed in step (1) into host cells for expression; (3) Use TB medium, fermentation temperature at 30-40°C, and use 0.1-0.5mM when OD 600 reaches 1.5-2.0 IPTG is used for induction, and the induction time is 8-16h; (4) After the expressed target protein is prepared by affinity adsorption and purification on a nickel column, the endotoxin is removed by an endotoxin adsorption column (Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher). The 0.22 ⁇ m pore filter membrane removes residual bacteria.
  • the steps of expression and purification of the transthyretin protein include: constructing the pETx-rhaPBAD-ttr plasmid (the entire nucleic acid sequence of the plasmid is shown in SEQ ID NO: 3) Transform into E.coli BL21(DE3) cells, culture the obtained recombinant E.coli BL21(DE3) in LB medium, prepare seed solution, and then insert it into 5L TB medium at 5% inoculum, temperature At 37°C, the rotating speed of the stirring blade was 150 rpm, and the culture was cultured to an OD 600 of 1.5-2.0; 0.4-2% rhamnose was added to induce 16-20 hours.
  • Endotoxin adsorption column (Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher) was used to remove endotoxin, and residual bacterial cells were removed through a 0.22 ⁇ m pore filter membrane.
  • carboxymethyl cellulose or its salts such as sodium carboxymethyl cellulose
  • chondroitin sulfate or its salts such as chondroitin sulfate A sodium salt
  • dextran such as dextran 70
  • ocular diseases related to ocular angiogenesis and/or ocular retinal leakage For example, diabetic retinopathy, age-related macular degeneration, and/or retinopathy of prematurity can be treated with synergistic treatment, and finally a better treatment effect can be obtained.
  • the fifteenth aspect of the present invention also provides an ophthalmic preparation, which comprises transthyretin and pharmaceutically acceptable excipients selected from "carboxymethyl cellulose or its salts, its Solvate, solvate of its pharmaceutically acceptable salt, or, crystal form", “chondroitin sulfate or its salt, solvate, solvate of its pharmaceutically acceptable salt, or, Its crystal form", “dextran or its salt, its solvate, its pharmaceutically acceptable salt solvate, or, its crystal form", and, “hyaluronic acid or its salt, its solvate, One or more of the solvate of its pharmaceutically acceptable salt, or its crystal form.
  • pharmaceutically acceptable excipients selected from "carboxymethyl cellulose or its salts, its Solvate, solvate of its pharmaceutically acceptable salt, or, crystal form", “chondroitin sulfate or its salt, solvate, solvate of its pharmaceutically acceptable salt, or, Its crystal form", “dextran or its salt, its solv
  • the carboxymethyl cellulose or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form has a viscosity of 800-1200 CP.
  • the chondroitin sulfate is chondroitin sulfate A.
  • the dextran is dextran 70.
  • the molecular weight of the dextran 70 is usually in the range of 64000-76000.
  • the molecular weight of the hyaluronic acid is 10,000 to 500,000.
  • the "salt" in the "carboxymethyl cellulose or its salt” can be a sodium salt or a calcium salt, for example, sodium carboxymethyl cellulose.
  • the "salt" in the "chondroitin sulfate or its salt” can be a sodium salt or a calcium salt, for example, chondroitin sulfate A sodium salt.
  • the concentration of the carboxymethyl cellulose or a salt thereof, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystal form thereof is 0-8 mg /mL but not 0 (ie 0 ⁇ concentration ⁇ 8mg/mL), preferably 2, 4, 6, or 8mg/mL.
  • the concentration of the chondroitin sulfate or a salt thereof, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystal form thereof is 0-40 mg/mL But it is not 0 (that is, 0 ⁇ concentration ⁇ 40 mg/mL), preferably 10, 20, 30, or 40 mg/mL.
  • the concentration of the dextran or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form is 0-0.8 mg/mL but It is not 0 (that is, 0 ⁇ concentration ⁇ 0.8 mg/mL), preferably 0.2, 0.4, 0.6, or 0.8 mg/mL.
  • the content of the hyaluronic acid or its salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystal form in mass volume percentage is less than or equal to 6% , Preferably 1-4%, more preferably 2%.
  • the transthyretin is as shown in (a), (b) or (c):
  • (b) A protein derived from (a) that has the function of inhibiting angiogenesis as shown in the amino acid sequence of (a) by substitution, deletion or addition of one or more amino acids;
  • (c) A protein represented by a sequence in which the amino acid sequence in (a) or (b) has been hydrophilically modified or hydrophobically modified.
  • amino acid sequence of the protein derived from (a) may be as shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
  • the hydrophilic modification or hydrophobic modification may be performed on the cysteine at position 10 in the amino acid sequence of (a), preferably the amino acid in (a)
  • the modification is carried out using a long-chain hydrophobic fragment such as n-dodecane on the cysteine at position 10 in the sequence, or, by using the cysteine at position 10 in the amino acid sequence in (a) Maleimide is connected to n-dodecane for the modification.
  • the content of the transthyretin contained in the ophthalmic preparation is 4-30 ⁇ mol/L, preferably 5-30 ⁇ mol/L, more preferably 10-20 ⁇ mol/L, such as 10, 15 or 20 ⁇ mol/L .
  • the ophthalmic preparation may also contain other pharmaceutically acceptable excipients conventionally used in the art, such as physiological saline.
  • physiological saline the usage amount of the physiological saline can be used in accordance with the standards stipulated in the field.
  • the ophthalmic preparation may be in the form of a product conventionally applied to the eye in the art, for example, it may be a drop, such as eye drops, or a spray, a gel, or an ophthalmic liposome, etc. .
  • the ophthalmic preparation is an ophthalmic preparation that inhibits ocular retinal leakage and/or reduces the number of retinal neovascularization; the ophthalmic preparation is preferably for the treatment of diabetic retinopathy, age-related macular degeneration and/or premature infants Ophthalmic preparations for retinopathy.
  • the ophthalmic preparation is administered 1-3 times a day, and each application amount is preferably 0.3-0.8 nmol protein/eye.
  • the ophthalmic preparation is administered twice a day, 1 drop each time, for 3 months.
  • the ophthalmic preparation is administered once a day, 1 drop each time, for 5 days.
  • the ophthalmic preparation is administered twice a day, 1 drop each time, for 2 weeks.
  • the ophthalmic preparation is composed of 10 ⁇ mol/L transthyretin, 6 mg/mL sodium carboxymethyl cellulose and physiological saline.
  • the ophthalmic preparation is composed of 10 ⁇ mol/L transthyretin, 0.4 mg/mL dextran 70 and physiological saline.
  • the ophthalmic preparation is composed of 10 ⁇ mol/L transthyretin, 20 mg/mL chondroitin sulfate A sodium salt and physiological saline.
  • the drops are composed of 10 ⁇ mol/L transthyretin, 2% hyaluronic acid and physiological saline.
  • the inventors also unexpectedly discovered during the experiment that when transthyretin and/or fusion protein are co-administered to the eye with specific compounds, the compound can interact with transthyretin and/or fusion protein. Synergistic cooperation and joint action can effectively treat or alleviate ocular diseases related to ocular retinal angiogenesis and/or ocular retinal leakage, so that the treatment effect is better.
  • the sixteenth aspect of the present invention also provides an ophthalmic preparation, which comprises a compound, its pharmaceutically acceptable salt, its glycoside, its solvate, its pharmaceutically acceptable salt solvate, or its crystal Type, and transthyretin; the compound is selected from one or more of diclofenac, vitamin A and luteolin.
  • the pharmaceutically acceptable salt is a sodium salt, such as diclofenac sodium.
  • the glycoside is luteolin.
  • the vitamin A is vitamin A1 and/or vitamin A2.
  • the vitamin A can be purchased from China National Medicines, with the national medicine code of CATOCCHM700908, CAS: 68-26-8.
  • the content of transthyretin is 4-30 ⁇ mol/L, preferably 5-30 ⁇ mol/L, more preferably 10-20 ⁇ mol/L, such as 10, 15 or 20 ⁇ mol/L .
  • the content of diclofenac (or, for example, its sodium salt diclofenac sodium) is 5-20 ⁇ mol/L, for example, 10 ⁇ mol/L.
  • the content of vitamin A in the ophthalmic preparation is 2-10 ⁇ mol/L, for example, 5 ⁇ mol/L.
  • the content of the luteolin is 2-10 ⁇ mol/L, for example, 5 ⁇ mol/L.
  • the transthyretin is as shown in (a), (b) or (c):
  • (b) A protein derived from (a) that has the function of inhibiting angiogenesis as shown in the amino acid sequence of (a) by substitution, deletion or addition of one or more amino acids;
  • (c) A protein represented by a sequence in which the amino acid sequence in (a) or (b) has been hydrophilically modified or hydrophobically modified.
  • amino acid sequence of the protein derived from (a) may be as shown in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.
  • the hydrophilic modification or hydrophobic modification is performed on the cysteine at position 10 on the amino acid sequence in (a), preferably on the amino acid sequence in (a)
  • the modification is carried out by using a long-chain hydrophobic fragment such as n-dodecane on the cysteine at position 10, or, by using the cysteine at position 10 on the amino acid sequence in (a).
  • the imide is linked to n-dodecane for the modification.
  • the ophthalmic preparation also contains pharmaceutically acceptable excipients such as physiological saline.
  • pharmaceutically acceptable excipients such as physiological saline.
  • the usage amount of the physiological saline can be used in accordance with the standards stipulated in the field.
  • the ophthalmic preparation also contains a pharmaceutically acceptable excipient such as a surfactant, the surfactant is, for example, Tween 80, and its content is preferably 5% (v/v).
  • a pharmaceutically acceptable excipient such as a surfactant
  • the surfactant is, for example, Tween 80, and its content is preferably 5% (v/v).
  • the ophthalmic preparation also contains pharmaceutically acceptable excipients, and the excipients are sodium carboxymethyl cellulose, dextran 70, chondroitin sulfate A sodium salt or hyaluronic acid.
  • the concentration of the sodium carboxymethyl cellulose is 0-8 mg/mL but not 0 (ie, 0 ⁇ concentration ⁇ 8 mg/mL), preferably 2, 4, 6 or 8 mg/mL.
  • the concentration of the dextran 70 is 0-0.8mg/mL but not 0 (ie 0 ⁇ concentration ⁇ 0.8mg/mL), preferably 0.2, 0.4, 0.6 or 0.8mg/mL.
  • the concentration of the chondroitin sulfate A sodium salt is 0-40 mg/mL but not 0 (ie, 0 ⁇ concentration ⁇ 40 mg/mL), preferably 10, 20, 30, or 40 mg/mL.
  • the content of the hyaluronic acid in mass volume percentage is preferably ⁇ 6%, preferably 1-4%, and more preferably 2%.
  • the ophthalmic preparation may be in the form of a product conventionally applied to the eye in the art, for example, it may be a drop, such as eye drops, or a spray, a gel, or an ophthalmic liposome, etc. .
  • the ophthalmic preparation is administered 1-3 times a day, and each application amount is preferably 0.3-0.8 nmol protein/eye.
  • the ophthalmic preparation is administered twice a day, 1 drop each time, for 3 months.
  • the ophthalmic preparation is administered once a day, 1 drop each time, for 5 days.
  • the ophthalmic preparation is administered twice a day, 1 drop each time, for 2 weeks.
  • the ophthalmic preparation is composed of 10 ⁇ mol/L transthyretin, excipients, 10 ⁇ mol/L diclofenac sodium and physiological saline, and the excipients are 6 mg/mL sodium carboxymethyl cellulose, 0.4 Any one of mg/mL dextran 70, 20 mg/mL chondroitin sulfate A sodium salt, and 2% (mass volume percentage) hyaluronic acid.
  • the ophthalmic preparation is composed of 10 ⁇ mol/L transthyretin, excipients, 5 ⁇ mol/L vitamin A, 5% (v/v) Tween 80 and physiological saline, and the excipients are Any one of 6 mg/mL sodium carboxymethyl cellulose, 0.4 mg/mL dextran 70, 20 mg/mL chondroitin sulfate A sodium salt, and 2% (mass volume percentage) hyaluronic acid.
  • the ophthalmic preparation is composed of 10 ⁇ mol/L transthyretin, excipients, 5 ⁇ mol/L luteolin and physiological saline, and the excipients are 6 mg/mL sodium carboxymethyl cellulose, Any one of 0.4 mg/mL dextran 70, 20 mg/mL chondroitin sulfate A sodium salt, and 2% (mass volume percentage) hyaluronic acid.
  • the ophthalmic preparation is an ophthalmic preparation that inhibits ocular retinal leakage and/or reduces the number of retinal neovascularization, and the ophthalmic preparation is preferably used to treat diabetic retinopathy, age-related macular degeneration and/or premature infants Ophthalmic preparations for retinopathy.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the pharmaceutically acceptable excipients and the like can usually be obtained commercially, for example, from Sinopharm Group.
  • the mentioned compounds can usually be obtained commercially, for example, from Sinopharm Group, Bailingwei Technology Co., Ltd. and the like.
  • the "include or include” may mean that in addition to the ingredients listed below, there are other ingredients; in some cases, it may also mean “consisting of”, that is, only including the ingredients listed below. No other ingredients are present in the listed ingredients.
  • amino acids in the present invention are conventional in the art unless otherwise specified.
  • the amino acids corresponding to the specific abbreviations are shown in Table 1-1.
  • Amino acid name Three letter symbol Single letter symbol Amino acid name Three letter symbol Single letter symbol Amino acid name Three letter symbol Single letter symbol Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine (phenylalanine) Phe F Cysteine Cys C Proline Pro P Glutamine (glutanine) Gln Q Serine Ser S Glutamicacid Glu E Threonine Thr T Glicine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y
  • codons corresponding to the amino acids are also conventional in the art, and the corresponding relationships between specific amino acids and codons are shown in Table 1-2.
  • transthyretin has better biocompatibility and safety in the human body, not only can it pass through the ocular barrier, but also can effectively deliver foreign proteins and/or polypeptides to the eyes. Inside, it has the effect of curing ocular diseases and has important application prospects in the field of medicine as a substitute for injection drugs.
  • transthyretin when transthyretin is used to treat ocular diseases such as DR, AMD, ROP, etc., transthyretin can cross the corneal barrier and enter the vitreous and fundus, which significantly inhibits Eyeball retinal leakage can significantly reduce the number of retinal neovascularization, and effectively alleviate the pathological phenomena of DR, AMD, ROP and other eye diseases.
  • FIG 1 shows the three-dimensional structure of Transthyretin (TTR) (PDB ID: 1ICT).
  • FIG. 2 shows that the hydrophobic domain of TTR core carries strong hydrophobic thyroxine molecules across various cells.
  • Figure 3 shows the amino acid sequence similarity between human TTR and TTR derived from SD rats, C57BL/6 mice, and rabbits is >95%. Among them, the amino acid sequence of TTR derived from SD rats is shown in SEQ ID NO: 9. The amino acid sequence of TTR derived from C57BL/6 mouse is shown in SEQ ID NO: 10.
  • Figure 4 is a plasmid map of pET21a(+)-His-tag-TTR-X.
  • the front end of the gene sequence is fused to express the His-tag sequence, and it is connected to the plasmid through two restriction enzyme cleavage sites, Nde I and EcoR I; "X" indicates a protein fused with TTR.
  • Figure 5 shows the electrophoresis pattern of E. coli BL21(DE3) expressing human transthyretin, the purified product of fusion protein and green fluorescent protein, egg white lysozyme and egg white albumin standard products.
  • Figure 6 shows the content of TTR in cornea, vitreous and fundus samples (retina, choroid) of C57BL/6 mice and SD rats after TTR drops.
  • Figure 7 is a western-blot map of the target protein in the vitreous cavity of human-derived transthyretin and its fusion protein in rats and rabbits after two weeks of instillation.
  • the left eye is the instillation eye and the right eye is the control eye.
  • the vitreous sample is detected by anti-TTR antibody for the presence of background TTR positive signal; and when the anti-His-tag antibody is used for detection, the drop
  • the positive signal in the eye increased significantly, indicating that human TTR can effectively enter the eye to reach the vitreous; TTR and GFP, Lysozyme, Ovalbumin and other foreign proteins can effectively enter the eye after fusion expression, but the above-mentioned proteins that are not fused and expressed cannot enter the eye .
  • Figure 8 shows the number of retinal leakage and retinal neovascularization in SD rats after STZ induced by TTR eye drops.
  • FIG. 9 shows that TTR eye drops prevented the ROP modeling process.
  • FIG. 10 shows that TTR eye drops inhibit the pathological process of ROP.
  • FIG 11 shows that TTR eye drops inhibited the pathological process of the AMD model.
  • Figure 12 shows the process of chemical modification of human TTR.
  • FIG. 13A shows that the protein structure is a TTR dimer.
  • the diclofenac ligand molecules are indicated by arrows.
  • One molecule of TTR dimer can bind two molecules of diclofenac.
  • Figure 13B shows the interaction of diclofenac with TTR amino acid residues.
  • Figure 14A shows the molecular simulation performed by Discovery studio software and found that vitamin A1 can stably bind to the hydrophobic channel of the TTR multimer.
  • the protein structure in the figure is TTR dimer.
  • the vitamin A1 ligand molecule is indicated by the arrow, and one molecule of TTR dimerizes.
  • the body can bind a molecule of vitamin A1.
  • Figure 14B shows the interaction of vitamin A1 with TTR amino acid residues.
  • FIG 15A shows the molecular simulation performed by Discovery studio software. It is found that vitamin A2 can stably bind to the hydrophobic channel of TTR multimer.
  • the protein structure in the figure is TTR dimer.
  • the vitamin A2 ligand molecule is indicated by the arrow, and one molecule of TTR dimerizes.
  • the body can bind a molecule of vitamin A2.
  • Figure 15B shows the interaction of vitamin A2 with TTR amino acid residues.
  • Figure 16A shows the molecular simulation performed by Discovery studio software and found that luteolin can stably bind to TTR multimers.
  • the protein structure in the figure is a TTR dimer.
  • the luteolin ligand molecule is indicated by an arrow, one molecule of TTR dimer. Can be combined with a molecule of luteolin.
  • Figure 16B shows the interaction of luteolin and TTR amino acid residues.
  • Figure 17A shows the molecular simulation performed by Discovery studio software and found that sulfamethoxazole can stably bind to TTR polymers.
  • the protein structure in the figure is TTR dimer.
  • the sulfamethoxazole ligand molecules are indicated by arrows, one molecule of TTR and two The polymer can be combined with one molecule of sulfamethoxazole.
  • Figure 17B shows the interaction of sulfamethoxazole with TTR amino acid residues.
  • TTR Transthyretin
  • the preparation of transthyretin includes the following steps:
  • pET 21a(+)-His-tag-TTR Synthetic nucleotide sequence of His-tag-TTR shown in SEQ ID NO: 4 (wherein the amino acid sequence of TTR used is shown in SEQ ID NO: As shown in 1, pET 21a was purchased from the ATCC China Culture Collection), and it was connected to pET 21a(+) with Nde I and EcoR I enzymes, and was verified by sequencing (the sequencing company is Nanjing GenScript Biotechnology Co., Ltd., Same), the construction is successful.
  • TTR Expression and purification of recombinant TTR:
  • the pET 21a(+)-His-tag-TTR plasmid constructed in step (1) is transformed into E.coli BL21(DE3) cells, and the obtained recombinant E.coli BL21(DE3) Cultivate in LB medium, prepare seed solution, and then add 5% inoculum to 5L TB medium, temperature 37°C, stirrer speed 150rpm, cultivate until OD 600 is 1.5-2.0; add 0.1-0.5 mM IPTG induced 8-16h (Table 1).
  • TTR was prepared by using high-pressure homogenization to break the bacteria and passing the supernatant through a nickel column for affinity adsorption.
  • the resulting protein is passed through an endotoxin adsorption column (Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher) to remove endotoxin, and a 0.22 ⁇ m pore filter membrane is used to remove residual bacterial cells.
  • the TTR protein production was tested, and the results are shown in Table 1. When the OD 600 is 1.5-1.8, with 0.3-0.5mM IPTG induced for 12-14h, the obtainable protein yield is ⁇ 17mg/g wet bacteria.
  • TTR-GFP transthyretin-green fluorescent protein fusion protein
  • TTR-GFP fusion protein Transform the recombinant plasmid constructed in step (1) into E.coli BL21(DE3) cells, and culture the obtained recombinant E.coli BL21(DE3) in LB medium, Prepare seed solution, with 5% inoculum, insert 5L TB medium, temperature 37°C, stir paddle speed 150rpm, cultivate OD 600 to 1.5-2.0; add 0.1-0.5mM IPTG for induction 8-16h; use high pressure The bacteria were homogenized and the supernatant was passed through a nickel column for affinity adsorption to prepare the TTR-GFP fusion protein.
  • the resulting protein is passed through an endotoxin adsorption column (Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher) to remove endotoxin, and a 0.22 ⁇ m pore filter membrane is used to remove residual bacterial cells.
  • the TTR-GFP protein production was tested, and the results are shown in Table 2. When the OD 600 is 1.5-1.9, with 0.3-0.5mM IPTG for 12h, the obtainable protein yield is ⁇ 10mg/g wet bacteria.
  • TTR-Lysozyme The fusion protein of transthyretin-egg white lysozyme (TTR-Lysozyme) is prepared as follows:
  • Cultivate coli BL21(DE3) in LB medium prepare seed solution, inoculate 5L TB medium with 5% inoculum, temperature 37°C, stirrer speed 150rpm, cultivate OD 600 to 1.5-2.0; add 0.1 -0.5mM IPTG for 8-16h induction; use high-pressure homogenization to break the bacteria and pass the supernatant through a nickel column affinity adsorption to prepare TTR-Lysozyme fusion protein.
  • the resulting protein is passed through an endotoxin adsorption column (Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher) to remove endotoxin, and a 0.22 ⁇ m pore filter membrane is used to remove residual bacterial cells.
  • endotoxin adsorption column Pieris TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher
  • a 0.22 ⁇ m pore filter membrane is used to remove residual bacterial cells.
  • transthyretin-egg albumin fusion protein (TTR-Ovalbumin) is prepared as follows:
  • TTR-Ovalbumin fusion protein the pET 21a(+)-His-tag-TTR-Ovalbumin plasmid constructed in step (1) was transformed into E.coli BL21(DE3) cells, and the resulting recombinant E. Cultivate coli BL21(DE3) in LB medium, prepare seed solution, inoculate 5L TB medium with 5% inoculum, temperature 37°C, stirrer speed 150rpm, cultivate OD 600 to 1.5-2.0; add 0.1 -0.5mM IPTG for 8-16h induction; use high-pressure homogenization to break the bacteria and pass the supernatant through the nickel column affinity adsorption to prepare the TTR-Ovalbumin fusion protein.
  • the resulting protein is passed through an endotoxin adsorption column (Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher) to remove endotoxin, and a 0.22 ⁇ m pore filter membrane is used to remove residual bacterial cells.
  • endotoxin adsorption column Pieris TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher
  • a 0.22 ⁇ m pore filter membrane is used to remove residual bacterial cells.
  • Figure 5 shows the electrophoresis patterns of E. coli BL21(DE3) expressing transthyretin, purified fusion protein products, green fluorescent protein, egg white lysozyme and egg white albumin standard products. It can be seen from the figure that the above-mentioned proteins are all correctly expressed.
  • TTR The amino acid sequence of TTR is shown in SEQ ID NO: 1), and the overall nucleic acid sequence of the obtained plasmid is shown in SEQ ID NO: 3. After sequencing verification (the sequencing company is Nanjing GenScript Biotechnology Co., Ltd.), the construction was successful.
  • the supernatant is prepared by nickel (Ni + ) column chromatography to obtain human TTR.
  • Endotoxin adsorption column (Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher) was used to remove endotoxin, and residual bacterial cells were removed through a 0.22 ⁇ m pore filter membrane.
  • the resulting human TTR protein yield is shown in Table 5. When the OD 600 is 1.8-2.0, with 1.6-2% rhamnose induced for 18-19 hours, the obtainable protein yield is ⁇ 50mg/g wet bacteria.
  • rat-derived TTR The expression and purification of recombinant rat-derived TTR and mouse-derived TTR are the same as described above, except that the human TTR optimized nucleic acid sequence is replaced with the corresponding murine TTR nucleic acid sequence, and the rest of the steps are the same.
  • the amino acid sequence of rat-derived TTR is shown in SEQ ID NO: 9
  • the amino acid sequence of mouse-derived TTR is shown in SEQ ID NO: 10.
  • Chemically modified human TTR design and synthesize a chemically modified group, which is a hydrophobic modified fragment formed by connecting maleimide, n-dodecane and 5-aminofluorescein (Ex 490nm, Em 520nm), and then perform targeted chemical modification of the chemical modification group with the only cysteine (C) residue in the recombinantly expressed and purified human TTR.
  • C cysteine residue
  • the human TTR and the chemical modification group react slowly with a 1:5 molar ratio (the reaction process is shown in Figure 12). After the reaction is terminated, the remaining chemical modification reagents are removed by ultrafiltration and the TTR is concentrated.
  • the sample is detected by a fluorescence spectrometer and excited at a wavelength of 490nm with an emission wavelength of 520nm, indicating that the only cysteine residue of TTR has been successfully analyzed.
  • Targeted modification named human TTR-Modified.
  • the T7 promoter on the reconstructed pET-21a plasmid in Example 5 was replaced by the rhaPBAD promoter (rhamnodon inducible type), and was ligated with human TTR with unoptimized nucleic acid sequence to construct a recombinant plasmid pETx- rhaPBAD-ttr (not optimized), that is, the difference between the obtained recombinant plasmid and the recombinant plasmid pETx-rhaPBAD-ttr in Example 5 is only whether the TTR is optimized.
  • the recombinant human TTR was expressed and purified using the same method as in Example 5, part (2), and the results are shown in Table 6 below.
  • the second part TTR itself and after fusion with protein can enter the vitreous cavity and fundus through the corneal barrier
  • Example 7 Human-derived TTR eye drops across the corneal barrier into the vitreous cavity and fundus
  • the human TTR obtained in Example 5 was configured to 10 ⁇ mol/L (containing physiological saline), and the C57BL/6 mice (purchased from Shanghai Experimental Animal Research Center, 8 weeks old) and SD rats (purchased from Shanghai Experimental Animal Research Center, 8 weeks of age) gave an eye drop, instilled one drop ( ⁇ 30 ⁇ l), waited for 3 hours, and then sacrificed. The cornea, vitreous and fundus samples (retina, choroid) were taken out, and the proteins were extracted separately. Because the recombinant human TTR has a His-tag tag, the rabbit anti-His-tag antibody is used as the primary antibody and the donkey anti-rabbit antibody is used as the secondary antibody.
  • the human TTR obtained in Example 5 is configured to 5-30 ⁇ mol/L (containing physiological saline, and 0-6% low molecular weight hyaluronic acid), respectively for C57BL/6 mice (8 weeks old) and SD Rats (8 weeks old) were given eye drops and sacrificed after 3-72h.
  • the vitreous and fundus samples were taken to extract protein.
  • Rabbit anti-His-tag antibody was used as the primary antibody, and donkey anti-rabbit antibody was used as the secondary antibody.
  • the determination was carried out by ELISA.
  • the human TTR and human TTR-Modified obtained in Example 5 are configured to 10 ⁇ mol/L (containing physiological saline, and 2% low molecular weight hyaluronic acid), respectively for C57BL/6 mice (8 weeks old) And SD rats (8 weeks old) were instilled and sacrificed after 3-72h.
  • the vitreous and fundus samples (retina, choroid) were taken to extract protein, rabbit anti-His-tag antibody was used as the primary antibody, and donkey anti-rabbit antibody was used as the primary antibody.
  • the secondary antibody was used to determine the content of human TTR in the vitreous and fundus samples of C57BL/6 mice and SD rats by ELISA. The results showed that the efficiency of human TTR-Modified into the vitreous and fundus after being modified by the long hydrophobic fragment was significantly higher than that of unmodified human TTR (Table 7-2).
  • Example 8 TTR eye drops across the corneal barrier into the vitreous cavity
  • the purified TTR protein (at a concentration of 4 ⁇ mol/L) prepared in Example 1 after removal of endotoxin and bacteria was treated with eye drops in healthy SD rats (rattus norregicus) (6 weeks old) and New Zealand big-eared rabbits ( Oryctolagus cuniculus) (2 months old, ⁇ 2.5kg), the left eye was dripped with protein sample, and the right eye was dripped with saline as a blank control.
  • the number of drops per day is 1-3 times, and the amount of each drop is 0.4-0.8nmol. Two weeks later, the eyeball was removed and the vitreous sample was obtained.
  • TTR can enter the vitreous cavity from the ocular surface.
  • the western-blot result with Anti-His tag antibody as the primary antibody showed that SD rats The signal intensity of the exogenous TTR in the vitreous of the left eye is 28.7 times that of the right eye, and the signal intensity of the exogenous TTR in the vitreous of the left eye of New Zealand big-ear rabbits is 35.6 times that of the right eye; the anti-His tag antibody is used as the primary antibody to determine The content of TTR in the vitreous sample (Table 8, 9). The results showed that after adding 0.6nmol twice a day, higher levels of exogenous TTR were detected in the vitreous of SD rats and New Zealand rabbits.
  • Figure 3 shows that human/rat/mouse/rabbit-derived transthyretin has been homologously aligned, and the sequence similarity reaches nearly 95%. It can be seen that the positive signal of the body TTR in Figure 7A is intraocular Homologous protein signal.
  • Example 9 TTR-GFP fusion protein crosses the corneal barrier into the vitreous cavity by eye drops
  • TTR-GFP protein concentration 4 ⁇ mol/L prepared in Example 2 after removal of endotoxin and bacteria was treated with eye drops in healthy SD rats (rattus norregicus) (6 weeks old) and New Zealand large ears Rabbit (Oryctolagus cuniculus) (2 months old, ⁇ 2.5kg), the left eye is the eye with the protein sample dripped, and the right eye is dripped with physiological saline as the blank control.
  • the number of drops per day is 1-3 times, and the amount of each drop is 0.4-0.8nmol. Two weeks later, the eyeball was removed and a vitreous sample was obtained.
  • TTR-GFP can enter the vitreous cavity from the ocular surface.
  • the western-blot with Anti-GFP antibody as the primary antibody showed that the SD was large.
  • the signal intensity of exogenous GFP in the vitreous of the left eye of mice is 62.3 times that of the right eye, and the signal intensity of exogenous GFP in the vitreous of the left eye of New Zealand big-ear rabbits is 47.6 times that of the right eye; western-blot with Anti-Histag antibody as the primary antibody
  • the results showed that the signal intensity of exogenous TTR in the vitreous of the left eye of SD rats was stronger but there was no signal in the right eye.
  • the signal intensity of exogenous TTR in the vitreous of the left eye of New Zealand bull rabbits was 45.4 times that of the right eye.
  • the content of TTR-GFP in the vitreous sample was determined by the ELISA method with Anti-His tag antibody as the primary antibody (Table 10, 11). The results showed that after adding 0.6nmol twice a day, higher levels of exogenous TTR-GFP were detected in the vitreous of SD rats and New Zealand rabbits.
  • TTR-Lysozyme fusion protein crosses the corneal barrier into the vitreous cavity by eye drops
  • TTR-Lysozyme protein (concentration 4 ⁇ mol/L) prepared in Example 3 after removal of endotoxin and bacteria was treated with eye drops in healthy SD rats (rattus norregicus) (6 weeks old) and New Zealand large ears Rabbit (Oryctolagus cuniculus) (2 months old, ⁇ 2.5kg), the left eye is the eye with the protein sample dripped, and the right eye is dripped with physiological saline as the blank control.
  • the number of drops per day is 1-3 times, and the amount of each drop is 0.4-0.8nmol. Two weeks later, the eyeball was removed and a vitreous sample was obtained.
  • the signal intensity of the exogenous Lysozyme in the vitreous of the left eye of New Zealand big-ear rabbits is stronger but there is no signal in the right eye; western- with Anti-His tag antibody as the primary antibody Blot results showed that the signal intensity of exogenous TTR in the vitreous of the left eye of SD rats was 30.2 times that of the right eye, and the signal intensity of exogenous TTR in the vitreous of the left eye of New Zealand big-ear rabbits was 46.3 times that of the right eye.
  • the content of TTR-Lysozyme in the vitreous sample was determined by ELISA with Anti-His tag antibody as the primary antibody (Table 12, 13). The results showed that after the 0.6nmol content was dropped twice a day, high levels of exogenous TTR-Lysozyme were detected in the vitreous of SD rats and New Zealand rabbits.
  • Example 11 TTR-Ovalbumin fusion protein crosses the corneal barrier into the vitreous cavity by eye drops
  • TTR-Ovalbumin protein (concentration 4 ⁇ mol/L) prepared in Example 4 after removal of endotoxin and bacteria was treated with eye drops in healthy SD rats (rattus norregicus) (6 weeks old) and New Zealand large ears Rabbit (Oryctolagus cuniculus) (2 months old, ⁇ 2.5kg), the left eye is the eye with the protein sample dripped, and the right eye is dripped with physiological saline as the blank control.
  • the number of drops per day is 1-3 times, and the amount of each drop is 0.4-0.8nmol. Two weeks later, the eyeball was removed and a vitreous sample was obtained.
  • the signal intensity of the exogenous TTR in the vitreous of the left eye of the New Zealand big-eared rabbit was stronger but there was no signal in the right eye.
  • the content of TTR-Ovalbumin in the vitreous sample was determined by the ELISA method with Anti-His tag antibody as the primary antibody (Table 14, 15). The results showed that after dropping 0.6nmol twice a day, high levels of exogenous TTR-Ovalbumin were detected in the vitreous of SD rats and New Zealand rabbits.
  • the third part TTR treatment of ocular diseases such as sugar network
  • Example 12 Treatment of DR (diabetic retinopathy) SD rats with human TTR eye drops
  • DR SD rats 8-week-old SD rats weighing 200-250g, fasting for 12-18h, intraperitoneally injecting 2% STZ (60mg/kg), cutting their tails and collecting blood after 48h and 72h, the blood glucose test paper test is higher than 16.7mM, the model is successful.
  • STZ 60mg/kg
  • the blood glucose test paper test is higher than 16.7mM
  • the model is successful.
  • DR SD rats are divided into 2 major groups, one group is DR SD rats without any treatment (5), and the other group is the human TTR eye drop group prepared in Example 5.
  • each left eye Human TTR (5-20 ⁇ mol/L) (normal saline + 2% hyaluronic acid) was dripped twice a day, and normal saline + 2% hyaluronic acid was dripped into the right eye; in addition, another group of normal SD rats ( No eye drops) served as a normal control group (5 animals).
  • TTR (10 ⁇ mol/L) eye drops were performed on the modeled ROP suckling mice for 5 days. Comparing the ROP and ROP/TTR (modeling) groups, in the later stage of the experiment (5 days), after the malformed neovascularization in the ROP group filled the retina, a large number of leakage areas appeared, and TTR eye drops can reverse this trend (Figure 10) ).
  • TTR 10 ⁇ mol/L TTR has the best effect (Table 17 ).
  • Example 14 Treatment of AMD (age-related macular degeneration) C57BL/6 mice with human TTR eye drops
  • the retina was photocoagulated with a krypton laser (647nm), power 360mW, diameter 50 ⁇ m, time 0.05s, 8 photocoagulation points per eye, induced choroidal neovascularization, and gradually proliferated to the retina Migrate to obtain AMD C57BL/6 mice.
  • AMD C57/BL6 mice were divided into an eye drop group and a non-eye drop group. There were 14 eyes in the eye drop group.
  • TTR was applied to the right eye. 5-20 ⁇ mol/L of the TTR prepared in Example 5 was added daily to the right eye.
  • TTR 10 ⁇ mol/L
  • Table 18 The pathological conditions of AMD in C57BL/6 mice induced by laser retinal photocoagulation with human TTR
  • TTR eye drops can effectively treat the pathological states of DR, AMD, and ROP in animal models of DR, AMD, and ROP.
  • Example 15 Human-derived TTR/rat-derived TTR eye drops to treat DR (diabetic retinopathy) SD rats 8 weeks old SD rats, weighing 200-250g, fasting for 12-18h, intraperitoneal injection of 2% STZ (60mg /kg), after 48h and 72h, the tail was cut and blood was collected. The blood glucose test paper test was higher than 16.7mM. The model was successfully established and DR SD rats were obtained. DR SD rats are divided into 5 major groups.
  • DR diabetic retinopathy
  • Group 1 is DR SD rats without any treatment (5);
  • Group 1 is the human TTR eye drop group prepared in Example 5, 5 rats, left eye daily 2 drops of human TTR (10 ⁇ mol/L) (normal saline + 2% hyaluronic acid), the right eye drops of normal saline + 2% hyaluronic acid;
  • 1 group is the rat-derived TTR prepared in Example 5 In the eye drop group, 5 eyes, the left eye was dropped twice a day with the rat-derived TTR (10 ⁇ mol/L) prepared in Example 5 (normal saline + 2% hyaluronic acid), and the right eye was dropped with normal saline + 2 % Hyaluronic acid;
  • 1 group is the human TTR-CL eye drop group prepared in Example 5, with 5 eyes, the left eye was instilled twice a day with the human TTR-CL prepared in Example 5 (10 ⁇ mol/ L) (normal saline + 2% hyaluronic acid), the right eye is dripped with s
  • Example 16 Human-derived TTR/rat-derived TTR eye drops to treat ROP (retinopathy of prematurity) SD rats
  • Example 17 Treatment of AMD (age-related macular degeneration) C57BL/6 mice with human TTR/mouse TTR eye drops
  • AMD C57/BL6 mice were divided into an eye drop group and a non-eye drop group.
  • the eye drop group was given TTR eye drops, and 10 ⁇ mol/L human TTR or mouse TTR (normal saline + 2% hyaluronic acid) was applied to the right eye every day.
  • Table 21 The pathological conditions of AMD in C57BL/6 mice induced by laser retinal photocoagulation with human TTR/mouse TTR
  • Example 18 Using sodium carboxymethyl cellulose as an adjuvant can promote TTR to pass through the corneal barrier
  • the human TTR prepared in Example 5 is configured to 5-30 ⁇ mol/L (containing physiological saline), and C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) are respectively used for eye drops After 3 hours, they were sacrificed. The vitreous and fundus samples were taken to extract the protein. The rabbit anti-His-tag antibody was used as the primary antibody, and the donkey anti-rabbit antibody was used as the secondary antibody. The vitreous body of C57BL/6 mice and SD rats was determined by ELISA. The amount of human TTR in the fundus sample.
  • the human TTR prepared in Example 5 is configured to 10 ⁇ mol/L (containing physiological saline and 0-8 mg/mL sodium carboxymethyl cellulose (purchased from Sinopharm, viscosity 800-1200CP)), respectively for C57BL /6 mice (8 weeks old) and SD rats (8 weeks old) were given eye drops, and they were sacrificed after 3-72h.
  • the vitreous body and fundus samples were taken to extract protein, and rabbit anti-His-tag antibody was used as the primary antibody.
  • the anti-rabbit antibody is a secondary antibody.
  • the content of human TTR in the vitreous and fundus samples of C57BL/6 mice and SD rats was determined by ELISA.
  • Example 19 Using dextran 70 as an adjuvant can promote TTR to pass through the corneal barrier
  • the human TTR prepared in Example 5 is configured to 5-30 ⁇ mol/L (containing physiological saline), and C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) are respectively used for eye drops After 3 hours, they were sacrificed. The vitreous and fundus samples were taken to extract the protein. The rabbit anti-His-tag antibody was used as the primary antibody, and the donkey anti-rabbit antibody was used as the secondary antibody. The vitreous body of C57BL/6 mice and SD rats was determined by ELISA. The amount of human TTR in the fundus sample.
  • the human TTR prepared in Example 5 is configured to 10 ⁇ mol/L (containing physiological saline and 0-0.8mg/mL dextran 70 (purchased from Sinopharm, molecular weight 64000-76000)), respectively, for C57BL/6 small Rats (8 weeks old) and SD rats (8 weeks old) were instilled into the eyes, and then sacrificed after 3-72 hours.
  • the vitreous and fundus samples were taken to extract protein.
  • Rabbit anti-His-tag antibody was used as primary antibody, donkey anti-rabbit antibody As the secondary antibody, the content of human TTR in the vitreous and fundus samples of C57BL/6 mice and SD rats was determined by ELISA.
  • Example 20 Using chondroitin sulfate A sodium salt as an excipient can promote TTR to pass through the corneal barrier
  • the human TTR prepared in Example 5 is configured to 5-30 ⁇ mol/L (containing physiological saline), and C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) are respectively used for eye drops After 3 hours, they were sacrificed. The vitreous and fundus samples were taken to extract the protein. The rabbit anti-His-tag antibody was used as the primary antibody, and the donkey anti-rabbit antibody was used as the secondary antibody. The vitreous body of C57BL/6 mice and SD rats was determined by ELISA. The amount of human TTR in the fundus sample.
  • the human TTR prepared in Example 5 is configured to 10 ⁇ mol/L (containing physiological saline and 0-40 mg/mL chondroitin sulfate A sodium salt (purchased from Sinopharm)), and the C57BL/6 mice (8 Week-old) and SD rats (8-week-old) were instilled in the eye, and then sacrificed after 3-72h.
  • the vitreous and fundus samples were taken to extract protein, rabbit anti-His-tag antibody was used as the primary antibody, and donkey anti-rabbit antibody was used as the secondary antibody , Determine the content of human TTR in the vitreous and fundus samples of C57BL/6 mice and SD rats by ELISA.
  • Example 21 Treatment of DR (diabetic retinopathy) SD rats with human TTR-adjuvant eye drops
  • DR SD rats 8-week-old SD rats weighing 200-250g, fasting for 12-18h, intraperitoneally injecting 2% STZ (60mg/kg), cutting their tails and collecting blood after 48h and 72h, the blood glucose test paper test is higher than 16.7mM, the model is successful.
  • STZ 60mg/kg
  • the blood glucose test paper test is higher than 16.7mM
  • the model is successful.
  • DR SD rats are divided into 6 groups, one group is DR SD rats without any treatment (5), and the other 5 groups are 5 rats/group.
  • the left and right eyes are each dripped twice a day, each time 30 ⁇ L, of which, the left eye is the human TTR eye drops prepared in Example 5, or the human TTR prepared in Example 5 plus excipients, specifically 10 ⁇ mol/L human TTR (physiological Saline solution), 10 ⁇ mol/L human TTR (physiological saline solution+6mg/mL sodium carboxymethylcellulose), 10 ⁇ mol/L human TTR (physiological saline solution+6mg/mL PEG400), 10 ⁇ mol/L human TTR ( Physiological saline solution + 0.4mg/mL dextran 70), 10 ⁇ mol/L human source TTR (physiological saline solution + 20mg/mL chondroitin sulfate A sodium salt) eye drops, the right eye uses physiological saline solution, or physiological saline solution with auxiliary materials Eye drops as a control, specifically using physiological saline solution, physiological saline solution + 6 mg/mL sodium carboxymethyl cellulose, physiological
  • Example 22 Treatment of AMD (age-related macular degeneration) C57BL/6 mice with human TTR-adjuvant eye drops
  • AMD C57BL/6 mice are divided into 6 groups, one group is AMD C57BL/6 mice without any treatment (5 mice), and the other 5 groups are 5 mice/group.
  • the left eye and the right eye each receive 2 drops a day Each time, 30 ⁇ L each time, of which, the left eye is the human TTR eye drops prepared in Example 5, or the human TTR prepared in Example 5 is used with excipients, specifically 10 ⁇ mol/L human eye drops are used.
  • Source TTR (physiological saline solution), 10 ⁇ mol/L human source TTR (physiological saline solution + 6 mg/mL sodium carboxymethyl cellulose), 10 ⁇ mol/L human source TTR (physiological saline solution + 6 mg/mL PEG400), 10 ⁇ mol/L Human TTR (physiological saline solution + 0.4mg/mL dextran 70), 10 ⁇ mol/L human TTR (physiological saline solution + 20mg/mL chondroitin sulfate A sodium salt) eye drops, the right eye uses physiological saline solution, or physiological Saline solution plus excipients for eye drops, as a control, specifically using physiological saline solution, physiological saline solution + 6mg/mL sodium carboxymethyl cellulose, physiological saline solution + 6mg/mL PEG400, physiological saline solution + 0.4mg/mL Dextran 70, physiological saline solution + 20mg/mL chondroitin s
  • mice Another group of normal C57BL/6 mice served as controls (5 mice). After 2 weeks of instillation, the animals were sacrificed, the retina was detached and EB staining was performed to observe the retinal vascular leakage, and Trypsin enzymatic hydrolysis to observe the neovascular density.
  • Suckling rats (SD rats) one week old were raised in a hyperbaric chamber, and the normal control group was raised in a normal environment (normal control group, 5). The hyperbaric oxygen chamber was raised for 5 days and then taken out to obtain ROP SD rats. All ROP SD rats together with the normal control group were kept in a normal environment for 5 days. During the 5 days, ROP SD rats were divided into 6 groups, 1 group was ROP SD mice without any treatment (5), and the other 5 groups were 5 rats/group. The left eye and the right eye were instilled daily.
  • Table 27 The pathological condition of human TTR/human TTR-adjuvant treatment of hyperbaric oxygen chamber-induced ROP in neonatal SD rats
  • Example 24 Computer simulation of the binding morphology of TTR and each ligand molecule
  • FIG. 13A the protein structure is a TTR dimer, and the diclofenac ligand molecules are indicated by arrows.
  • TTR dimer can bind two molecules of diclofenac.
  • FIG 13B The interaction of diclofenac with TTR amino acid residues is shown in Figure 13B.
  • vitamin A1 (retinol) can stably bind to the hydrophobic channel of the TTR polymer.
  • the protein structure shown in Figure 14A is a TTR dimer.
  • the vitamin A1 ligand molecule is indicated by an arrow, and one molecule of TTR dimer can bind one molecule of vitamin A1.
  • the interaction of vitamin A1 with TTR amino acid residues is shown in Figure 14B.
  • FIG. 15A The protein structure shown in Figure 15A is a TTR dimer.
  • the vitamin A2 ligand molecule is indicated by an arrow, and a molecule of TTR dimer can bind to a molecule of vitamin A2.
  • Figure 15B shows the interaction of vitamin A2 with TTR amino acid residues.
  • luteolin can bind stably to TTR polymer.
  • the protein structure shown in Figure 16A is a TTR dimer.
  • the luteolin ligand molecule is indicated by an arrow, and one molecule of TTR dimer can bind one molecule of luteolin.
  • the interaction of luteolin with TTR amino acid residues is shown in Figure 16B.
  • Example 25 Determination of dynamic specific binding parameters of TTR and each potential ligand molecule
  • Nano ITC was used to determine the affinity binding equilibrium dissociation constant K d between TTR and each ligand molecule or its salt mentioned in Example 24.
  • TTR solution 1000 ⁇ L
  • various ligand solutions of 100 ⁇ mol/L were added dropwise at a rate of min, and the affinity binding equilibrium dissociation constant K d was calculated using the built-in software (Table 28).
  • the affinity binding equilibrium dissociation constants of various ligand molecules with TTR are all close to 10 -8 mol/L, which is close to the ability of monoclonal antibodies to recognize a single epitope, indicating that the above ligand molecules can interact with TTR specifically recognizes and binds.
  • Example 26 Human TTR-ligand molecular complexes cross the corneal barrier and enter the vitreous and fundus
  • the human TTR prepared in Example 5 was configured to 10 ⁇ mol/L (containing physiological saline, 2% low molecular weight hyaluronic acid and 10 ⁇ mol/L diclofenac sodium/5 ⁇ mol/L vitamin A+5%(v/v) vomiting Temperature 80/5 ⁇ mol/L luteolin (addition ratio refers to the results of computer simulation in Example 24), C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) were given eye drops, after 3 -72h later, they were sacrificed. The vitreous and fundus samples were taken to extract protein. Rabbit anti-His-tag antibody was used as the primary antibody and donkey anti-rabbit antibody as the secondary antibody.
  • the vitreous body and fundus of C57BL/6 mice and SD rats were measured by ELISA.
  • the content of human-derived TTR in the sample Refer to the entry of diclofenac sodium eye drops on page 115 of the second part of the Chinese Pharmacopoeia 2020 edition to determine the content of diclofenac sodium in the sample; refer to the entry of vitamin A on page 1472 of the second part of the Chinese Pharmacopoeia 2020, Determine the content of vitamin A in the sample; refer to the British Pharmacopoeia BP2017, Luteolin-7-glucoside entry to determine the content of luteolin in the sample.
  • Example 27 Treatment of DR (diabetic retinopathy) SD rats by eye drops with human TTR-ligand molecular complex
  • DR SD rats 8-week-old SD rats weighing 200-250g, fasting for 12-18h, intraperitoneally injecting 2% STZ (60mg/kg), cutting their tails and collecting blood after 48h and 72h, the blood glucose test paper test is higher than 16.7mM, the model is successful.
  • STZ 60mg/kg
  • the blood glucose test paper test is higher than 16.7mM
  • the model is successful.
  • DR SD rats are divided into 5 groups, one group is DR SD rats without any treatment (5), and the other 4 groups are 5 rats/group.
  • the left and right eyes are each dripped twice a day, each time 30 ⁇ L, of which, the left eye is the human TTR eye drops prepared in Example 5, or the human TTR + ligand eye drops prepared in Example 5, specifically 10 ⁇ mol/L human TTR (physiological Saline solution + 2% hyaluronic acid), 10 ⁇ mol/L human source TTR (physiological saline solution + 2% hyaluronic acid + 10 ⁇ mol/L diclofenac sodium), 10 ⁇ mol/L human source TTR (physiological saline solution + 2% hyaluronic acid) +5 ⁇ mol/L vitamin A+5% Tween 80), 10 ⁇ mol/L human source TTR (physiological saline solution+2% hyaluronic acid+5 ⁇ mol/L luteolin) eye drops, the right eye uses physiological saline solution+2 % Hyaluronic acid, or physiological saline solution + 2% hyaluronic acid + ligand eye drops as a control, specifically
  • Example 28 Treatment of AMD (age-related macular degeneration) C57BL/6 mice by eye drops of human TTR-ligand molecular complex
  • the retina was photocoagulated with a krypton laser (647nm), power 360mW, diameter 50 ⁇ m, time 0.05s, 8 photocoagulation points per eye, induced choroidal neovascularization, and gradually proliferated to the retina Migrate to obtain AMD C57BL/6 mice.
  • AMD C57BL/6 mice are divided into 5 groups, one group is AMD C57BL/6 mice without any treatment (5 mice), and the other 4 groups are 5 mice/group, and the left eye and the right eye each receive 2 drops a day Each time, 30 ⁇ L each time, where the left eye is the human TTR eye drops prepared in Example 5, or the human TTR + ligand eye drops prepared in Example 5, specifically 10 ⁇ mol/L human Source TTR (physiological saline solution + 2% hyaluronic acid), 10 ⁇ mol/L human source TTR (physiological saline solution + 2% hyaluronic acid + 10 ⁇ mol/L diclofenac sodium), 10 ⁇ mol/L human source TTR (physiological saline solution + 2 % Hyaluronic acid+5 ⁇ mol/L vitamin A+5% Tween 80), 10 ⁇ mol/L human source TTR (physiological saline solution+2% hyaluronic acid+5 ⁇ mol/L luteolin) eye drops, the right eye uses physiological Saline solution
  • mice Another group of normal C57BL/6 mice served as controls (5 mice). After 2 weeks of instillation, the animals were sacrificed, the retina was detached and EB staining was performed to observe the retinal vascular leakage, and Trypsin enzymatic hydrolysis to observe the neovascular density.
  • Table 31 The pathological conditions of AMD in C57BL/6 mice induced by laser retinal photocoagulation by human TTR/human TTR-ligand molecular complex
  • Suckling rats (SD rats) one week old were raised in a hyperbaric chamber, and the normal control group was raised in a normal environment (normal control group, 5). The hyperbaric oxygen chamber was raised for 5 days and then taken out to obtain ROP SD rats. All ROP SD rats together with the normal control group were kept in a normal environment for 5 days. During the 5 days, ROP SD rats were divided into 5 groups, 1 group was ROP SD mice without any treatment (5), and the other 4 groups were 5 rats/group. The left eye and the right eye were instilled daily.
  • Comparative example 1 Human TTR uses different plasmids for recombinant expression
  • the supernatant is prepared by Ni+ column chromatography to obtain human TTR.
  • Endotoxin adsorption column (Pierce TM High Capacity Endotoxin Removal Spin Columns, ThermoFisher) was used to remove endotoxin, and residual bacterial cells were removed through a 0.22 ⁇ m pore filter membrane.
  • the resulting human TTR protein yield is shown in Table 33.
  • the specific implementation mode is the same as in Example 9, except that the GFP protein that is not fused with TTR is expressed according to the method of Example 2 (Genbank accession number is QAA95705.1), and the GFP protein that is not fused with TTR is subjected to the same operation steps as in Example 9.
  • An eye drop test was performed on SD rats and New Zealand big-eared rabbits, and the results showed that GFP did not enter the vitreous body of SD rats and New Zealand big-eared rabbits (Table 34, 35).
  • the specific implementation mode is the same as in Example 10, except that the Lysozyme protein (Genbank accession number AAL69327.1) that is not fused with TTR is expressed according to the method of Example 3, and the unfused Lysozyme protein is subjected to the same operation steps as in Example 10.
  • SD rats and New Zealand big-eared rabbits were tested by eye drops, and the results showed that Lysozyme did not enter the vitreous body of SD rats and New Zealand big-eared rabbits (Table 36, 37).
  • the specific implementation mode is the same as that in Example 11.
  • the difference is that the Ovalbumin protein (UniProt accession number P01012) that is not fused with TTR is expressed according to the method of Example 4, and the unfused Ovalbumin protein is subjected to the same operation steps as in Example 11. Rats and New Zealand big-eared rabbits were tested with eye drops, and the results showed that Ovalbumin did not enter the vitreous body of SD rats and New Zealand big-eared rabbits (Table 38, 39).
  • Table 38 The content of Ovalbumin in each drop is 0.6nmol, and the effect of Ovalbumin in the eyes of SD rats after different times a day
  • Table 39 The content of Ovalbumin in each drop of 0.6nmol, the effect of Ovalbumin in the eyes of New Zealand big-eared rabbits after different times of daily drop
  • the human TTR prepared in Example 5 is configured to 5-30 ⁇ mol/L (containing physiological saline), and C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) are respectively used for eye drops After 3 hours, they were sacrificed. The vitreous and fundus samples were taken to extract the protein. The rabbit anti-His-tag antibody was used as the primary antibody, and the donkey anti-rabbit antibody was used as the secondary antibody. The vitreous body of C57BL/6 mice and SD rats was determined by ELISA. The amount of human TTR in the fundus sample.
  • the human TTR prepared in Example 5 is configured to 10 ⁇ mol/L (containing physiological saline and 0-8mg/mL PEG400 (purchased from Sinopharm, molecular weight 360-440)), and the C57BL/6 mice (8 Week-old) and SD rats (8-week-old) were instilled in the eye, and then sacrificed after 3-72h.
  • the vitreous and fundus samples were taken to extract protein, rabbit anti-His-tag antibody was used as the primary antibody, and donkey anti-rabbit antibody was used as the secondary antibody , Determine the content of human TTR in the vitreous and fundus samples of C57BL/6 mice and SD rats by ELISA.
  • Example 26 The specific embodiments are the same as in Example 26, except that each ligand molecule that does not bind to TTR is performed on C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) according to the same operation steps as in Example 26. Eye drop test results showed that each ligand molecule alone could not enter the vitreous and fundus of C57BL/6 mice and SD rats (Table 41).
  • Table 42 shows the result data of the right eye after instillation. The results showed that eye drops of each ligand molecule alone could not improve the pathological condition of STZ-induced DR in SD rats (Table 42).
  • Table 43 shows the result data of the right eye after instillation. The results showed that eye drops of each ligand molecule alone could not improve the pathological condition of AMD in C57BL/6 mice induced by laser retinal photocoagulation (Table 43).
  • Table 44 shows the result data of the right eye after instillation. The results showed that eye drops of each ligand molecule alone could not improve the pathological condition of ROP in neonatal SD rats induced by hyperbaric oxygen chamber (Table 44).
  • Sulfamethoxazole has a broad antibacterial spectrum and strong antibacterial effect. It can hinder bacterial growth and is particularly effective against Staphylococcus and Escherichia coli. It is suitable for respiratory, urinary and intestinal infections; it is mainly used to treat avian cholera . It can be used to prepare eye drops such as compound sulfamethoxazole sodium eye drops (this product is a compound preparation, each 10 ml contains sulfamethoxazole sodium 400 mg, aminocaproic acid 200 mg, dipotassium glycyrrhizinate 10 mg, horse Chlorpheniramine chlorpheniramine 2 mg. It is mainly used for bacterial conjunctivitis, hordeolitis (stye) and bacterial blepharitis caused by sensitive bacteria.
  • eye drops such as compound sulfamethoxazole sodium eye drops (this product is a compound preparation, each 10 ml contains sulfame
  • FIG. 17A The protein structure shown in Figure 17A is a TTR dimer.
  • the sulfamethoxazole ligand molecule is indicated by an arrow.
  • One molecule of TTR dimer can bind one molecule of sulfamethoxazole.
  • the interaction of sulfamethoxazole with TTR amino acid residues is shown in Figure 17B.
  • Example 25 add 100 ⁇ mol/L sulfamethoxazole sodium salt solution dropwise at a rate of 1 ⁇ L/min to 10 ⁇ mol/L TTR solution (1000 ⁇ L), and use the built-in software to calculate the affinity binding equilibrium dissociation constant Kd is 7.03 ⁇ 10 -8 mol/L.
  • Example 26 Refer to the procedure in Example 26 and use TTR/sulfamethoxazole sodium to instill the eyes of rats and mice for 3-72 hours.
  • the corneas of rats and mice were damaged, namely, sulfamethoxazole sodium and TTR When used together, it will burn the cornea and is not biologically safe. It can be seen that after using Discoverystudio software molecular simulation to screen out ligands that are also biologically active, have anti-inflammatory effects and can bind to TTR, not all of them can improve the therapeutic effect when tested and verified.

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Abstract

本发明提供了转甲状腺素蛋白作为蛋白质类和/或多肽类药物通过眼部屏障进入眼内的载体方面的应用，所述转甲状腺素蛋白为由SEQ ID NO:1或其突变或其修饰所示氨基酸组成的蛋白质。还提供了转甲状腺素蛋白和/或转甲状腺素蛋白与药物的融合蛋白在制备滴剂中的应用以及一种滴剂，所述药物为蛋白质类和/或多肽类药物。转甲状腺素蛋白在人体内具有较好的生物相容性与安全性，能够有效地将外源蛋白和/或多肽输送到眼内，达到治疗眼部疾病的作用。

Description

转甲状腺素蛋白进入眼内以及在制备滴剂中的应用
本申请要求申请日为2019/12/17的中国专利申请201911302937.3、申请日为2020/6/5的中国专利申请202010506950.7、申请日为2020/9/16的中国专利申请202010976582.2、申请日为2020/9/16的中国专利申请202010974997.6的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及医药技术领域，具体涉及转甲状腺素蛋白作为蛋白质类和/或多肽类药物通过眼部屏障进入眼内的载体方面的应用，本发明还涉及转甲状腺素蛋白、和/或转甲状腺素蛋白与蛋白质类和/或多肽类药物组成的融合蛋白在制备滴剂中的应用，本发明进一步涉及一种含有转甲状腺素蛋白的眼用制剂及其制备方法以及在治疗与眼部血管新生和/或眼部视网膜渗漏等相关的眼部疾病中的应用。
背景技术
糖尿病视网膜病变(Diabetic Retinopathy，DR)简称“糖网”，是糖尿病微血管病的临床表现，也是糖尿病最严重的并发症之一，现在已经成为主要的致盲眼病之一。主要是在眼部长期高糖及缺氧的微环境下，逐步发生微血管阻塞、微血管瘤、出血、静脉扩张、黄斑水肿、新生血管、大量玻璃体出血、眼内纤维化、视网膜脱离等临床症状。
老年性黄斑变性(Age-related macular degeneration，AMD)，为黄斑区结构的衰老性改变。主要表现为视网膜色素上皮细胞对视细胞外节盘膜吞噬消化的能力下降，结果使未被完全消化的盘膜残余小体潴留于基底部细胞原浆中，并向细胞外排出，沉积于Bruch膜，形成玻璃膜疣。由于黄斑部结构与功能上的特殊性，此种改变更为明显。玻璃膜疣也见于正常视力的老年人，但由此继发的种种病理改变后，则导致黄斑部变性发生。或者引起Bruch膜断裂，脉络膜毛细血管通过破裂的Bruch膜进入RPE(retinal pigment epithelium，视网膜色素上皮)下及视网膜神经上皮下，形成脉络膜新生血管，脉络膜新生血管为视网膜下新生血管。由于新生血管壁的结构异常，导致血管的渗漏和出血，进而引发一系列的继发性病理改变。老年性黄斑变性大多发生于45岁以上，其患病率随年龄增长而增高，是当前老年人致盲的重要疾病。
早产儿视网膜病变(retinopathy of prematurity，ROP)是指在孕36周以下、低出生体重、长时间吸氧的早产儿，其未血管化的视网膜发生纤维血管瘤增生、收缩，并进一步 引起牵拉性视网膜脱离和失明。以往曾称为Terry综合征或晶状体后纤维增生症，但后者仅反映了该病的晚期表现。孕期更短或更低出生体重者，发生率可达60％～80％。其病因是由于早产儿出生后在温箱内高氧环境中过度吸氧，而移出温箱后处于相对缺氧的状态，未完全血管化的视网膜对氧产生血管收缩和血管增殖。
针对眼部疾病的治疗，需要将药物有效的传递到眼内，特别是上述所述的糖尿病视网膜病变、老年性黄斑变性以及早产儿视网膜病变等眼部疾病，以及色素性视网膜炎、青光眼导致的神经改变等其他眼部疾病；但由于眼部存在较多的屏障，将药物有效的传递到眼内充满了挑战性。因此，近来眼部给药系统受到了广泛的关注。
根据眼部解剖结构，眼部屏障主要包括了泪液屏障、角膜/结膜屏障、以及血眼屏障；多重屏障在保护眼部的同时，能够有效的阻挡外源化学及生物分子的侵入，但也给药物的传递带来了较大的阻碍。目前，眼部常用的给药方式主要包括常规滴眼、结膜下注射、巩膜给药，以及玻璃体内注射给药。其中，常规滴眼液与眼表接触时间较短，由于自身的结构与特性，一些外源药物及蛋白很难有效进入眼内；而结膜下注射、巩膜给药及玻璃体内注射通常会带来一定程度的创伤。视网膜激光治疗、玻璃体切除联合硅油填充术这些方法损伤较大，视力恢复不理想。
目前治疗DR、AMD以及ROP的最常规手段为玻璃体腔内注射抗vegf抗体，但是如上述所述这种方式通常会带来一定程度的创伤，多次给药必须有较长时间的间隔期。
目前，也有一些专利文献设计了能够有效突破各种眼部屏障到达眼内的功能性小肽，如郑颖，许讯等人的专利(一类新的抑制新生血管的小肽及其应用，CN2013100527142)，苏莉等人的专利(一类抑制新生血管的小肽及其应用，CN201310058978.9)，杨晓璐等人的专利(一种预防和/或治疗炎症反应的小分子多肽及其应用，CN201210581759.4)。
以上研究均存在一定的局限性，眼内注射会带来一定的创伤，而功能性小肽的输送效率较低半衰期较短，为此，有必要寻找更为安全、稳定、有效且对眼部无伤害或伤害较小的药物及给药方法。
发明内容
本发明为了克服现有技术中由于眼部屏障的存在而没有能够安全、稳定、有效且对眼部无伤害或伤害较小的将药物传递至眼内的给药方法和药物以治疗糖尿病视网膜病变(DR)、老年性黄斑变性(AMD)、早产儿视网膜病变(ROP)等眼部疾病的缺陷，提供了一种转甲状腺素蛋白(TTR)作为蛋白质类和/或多肽类药物通过眼部屏障进入眼内的载体方面的应用，一种转甲状腺素蛋白、和/或转甲状腺素蛋白与蛋白质类和/或多肽类药 物组成的融合蛋白在制备滴剂中的应用以及一种滴剂。
本发明人通过大量实验，意外发现通过使用滴剂进行滴眼而非本领域常规的进行注射的方式，转甲状腺素蛋白可有效跨越角膜屏障进入玻璃体及眼底，并且还能有效地输送外源蛋白和/或多肽，由此，可以根据眼部疾病的病理特征，选择合适的蛋白质类和/或多肽类药物，通过转甲状腺素蛋白输送至眼内，从而达到治疗眼部疾病的作用，在医药领域作为注射类药物的替代物方面具有重要的应用前景。
本发明人通过大量实验，还意外地发现在转甲状腺素蛋白跨越角膜屏障进入玻璃体及眼底之后，转甲状腺素蛋白可以显著抑制眼球视网膜渗漏，显著降低视网膜新生血管数，有效缓解DR、AMD、ROP等眼部疾病。即，无需与蛋白质类和/或多肽类药物融合，转甲状腺素蛋白本身即可治疗或缓解DR、AMD、ROP等眼部疾病，并且能够达到足够的治疗浓度和治疗时间(半衰期很长)。
而且，本领域技术人员仅知晓透明质酸具有湿润的作用，并不知晓其可以增加转甲状腺素蛋白的透过量，而本发明人还进一步发现在制备滴剂例如滴眼液时，使用透明质酸可以增加玻璃体腔内及眼底转甲状腺素蛋白的透过量，进而有效增加转甲状腺素蛋白的浓度。
为了解决上述技术问题，本发明第一方面提供了转甲状腺素蛋白作为蛋白质类和/或多肽类药物通过眼部屏障进入眼内的载体方面的应用，所述转甲状腺素蛋白如(a)、(b)或(c)所示：
(a)由SEQ ID NO:1所示氨基酸组成的蛋白质；
(b)在(a)中的氨基酸序列上经过取代、缺失或添加一个或几个氨基酸的序列所示的具有抑制血管新生功能的由(a)衍生的蛋白质；
(c)在(a)或(b)中的氨基酸序列上经过亲水性修饰或疏水性修饰的序列所示的蛋白质。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过22个氨基酸的取代。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过25个氨基酸的取代。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列的T3、T5、I26、N27、H31、R34、A36、A37、D38、D39、T40、S50、E61、E63、V65、I68、K70、I73、A81、H90、E92、P102、R104、T123、K126和/或E127发生取代。更佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过T3G、T5A、 I26V、N27D、H31K、R34K、A36T、D39G、T40S、S50A、E61D、E63K、V65T、I68V、K70R、I73L、H90Y、P102H、R104H、T123S、K126Q和E127N的取代。更佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过T3G、T5A、I26V、N27D、H31K、R34K、A36T、A37S、D38E、D39G、T40S、S50A、E61D、E63K、I68V、K70R、I73L、A81T、H90F、E92D、P102H、R104H、T123S、K126Q和E127N的取代。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过5个氨基酸的缺失。更佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列的第123-127位发生缺失。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质的氨基酸序列优选如SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11所示。
较佳地，所述(c)中，所述亲水性修饰或疏水性修饰在(a)中的氨基酸序列上的第10位的半胱氨酸上进行。较佳地，所述疏水性修饰为使用长链疏水片段例如正十二烷进行修饰。在本发明某一较佳实施例中，所述蛋白质为在(a)中的氨基酸序列上的第10位的半胱氨酸通过马来酰亚胺连接正十二烷修饰的序列所示的蛋白质。本发明中，所述的在(a)中的氨基酸序列上的第10位的半胱氨酸还可以是连接有5-氨基荧光素等荧光标记，以便追踪修饰后的蛋白。
较佳地，所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合表达；所述融合优选是将所述蛋白质类和/或多肽类药物融合在所述转甲状腺素蛋白的N端或C端。
较佳地，所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合是在微生物细胞中表达，并经过纯化。所述的微生物细胞可以为大肠杆菌，所述大肠杆菌包括但不限于E.coli BL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α、E.coli K12或E.coli TOP10。所述纯化优选是通过内毒素吸附柱(例如使用Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，再通过0.22μm孔径滤膜去除残留菌体。
较佳地，所述蛋白质类和/或多肽类药物包括但不限于：溶菌酶、白蛋白和/或EGFR抗体，且分子量不超过45kDa。其中，所述溶菌酶优选为鸡蛋清溶菌酶，其GenBank登录号为AAL69327.1。其中，所述白蛋白优选为鸡蛋清白蛋白。
较佳地，所述蛋白质类和/或多肽类药物包括治疗眼部视网膜渗漏和/或视网膜血管新生类眼部疾病例如糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的蛋白质类和/或多肽类药物。
较佳地，编码所述转甲状腺素蛋白的核苷酸序列如SEQ ID NO:2所示。
较佳地，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架质粒中的启动子为鼠李唐诱导型启动子，优选为rhaPBAD启动子。
较佳地，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架载体可以为pET-21a或与其具有25％及以上同源性的载体，所述与其具有25％及以上同源性的载体的序列优选如SEQ ID NO:8所示。
较佳地，表达所述转甲状腺素蛋白的重组质粒的核苷酸序列如SEQ ID NO:3所示。
较佳地，所述转甲状腺素蛋白可以是在微生物细胞中进行表达(形成转化体的形式)，并进一步可以经过纯化；所述的微生物细胞可以为大肠杆菌，所述大肠杆菌包括但不限于E.coli BL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α、E.coli K12或E.coli TOP10。所述纯化可以是通过内毒素吸附柱(例如使用Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，通过0.22μm孔径滤膜去除残留菌体。
较佳地，表达所述转甲状腺素蛋白时，通过培养包含所述转甲状腺素蛋白的基因的转化体至所得菌体的OD 600达到1.5-2.0(例如1.6、1.7、1.8或1.9)时进行表达。
较佳地，表达所述转甲状腺素蛋白时为使用诱导表达的试剂进行诱导表达，所述诱导表达的试剂的质量体积百分比为0.1-2％，例如0.2％、0.3％、0.4％、0.5％、0.7％、0.8％、1.2％或1.6％，所述诱导表达的时间优选为8-20h，例如10h、12h、14h、16h、17h、18h或19h；所述诱导表达的试剂优选为鼠李糖或者IPTG。
在本发明某一较佳实施例中，所述应用是将所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合表达所得的融合蛋白，通过滴眼的方式(例如制备成滴剂如滴眼液的形式)作用于眼睛，每日滴加次数为1-3次，每次滴加量为0.3-0.8nmol蛋白/眼。所述滴剂可以是以每日2次、每次1滴、持续3个月进行施用。所述滴剂可以是以每日1次、每次1滴、持续5天进行施用。所述滴剂可以是以每日2次、每次1滴、持续2周进行施用。
为了解决上述技术问题，本发明第二方面提供了转甲状腺素蛋白、和/或转甲状腺素蛋白与药物组成的融合蛋白在制备滴剂中的应用，所述药物为蛋白质类和/或多肽类药物，所述转甲状腺素蛋白如(a)、(b)或(c)所示：
(a)由SEQ ID NO:1所示氨基酸组成的蛋白质；
(b)在(a)中的氨基酸序列上经过取代、缺失或添加一个或几个氨基酸的序列所示的具有抑制血管新生功能的由(a)衍生的蛋白质；
(c)在(a)或(b)中的氨基酸序列上经过亲水性修饰或疏水性修饰的序列所示的蛋白质。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过22个氨基酸的取代。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过25个氨基酸的取代。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列的T3、T5、I26、N27、H31、R34、A36、A37、D38、D39、T40、S50、E61、E63、V65、I68、K70、I73、A81、H90、E92、P102、R104、T123、K126和/或E127发生取代。更佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过T3G、T5A、I26V、N27D、H31K、R34K、A36T、D39G、T40S、S50A、E61D、E63K、V65T、I68V、K70R、I73L、H90Y、P102H、R104H、T123S、K126Q和E127N的取代。更佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过T3G、T5A、I26V、N27D、H31K、R34K、A36T、A37S、D38E、D39G、T40S、S50A、E61D、E63K、I68V、K70R、I73L、A81T、H90F、E92D、P102H、R104H、T123S、K126Q和E127N的取代。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过5个氨基酸的缺失。更佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列的第123-127位发生缺失。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质的氨基酸序列优选如SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11所示。
较佳地，所述(c)中，所述亲水性修饰或疏水性修饰在(a)中的氨基酸序列上的第10位的半胱氨酸上进行。较佳地，所述疏水性修饰为使用长链疏水片段例如正十二烷进行修饰。在本发明某一较佳实施例中，所述蛋白质为在(a)中的氨基酸序列上的第10位的半胱氨酸通过马来酰亚胺连接正十二烷修饰的序列所示的蛋白质。本发明中，所述的在(a)中的氨基酸序列上的第10位的半胱氨酸还可以是连接有5-氨基荧光素等荧光标记，以便追踪修饰后的蛋白。
较佳地，所述融合蛋白的含量为4-30μmol/L，优选10-15μmol/L。
较佳地，所述转甲状腺素蛋白的含量为4-30μmol/L，优选5-30μmol/L，更优选10-20μmol/L，例如10、15、20μmol/L。
较佳地，所述滴剂中还含有生理盐水。本发明中，所述的生理盐水的使用量可按照本领域规定的标准进行使用即可。
较佳地，所述滴剂中还含有表面活性剂，所述表面活性剂例如为吐温80，其含量优选为5％(v/v)。
较佳地，所述滴剂中还含有透明质酸，所述透明质酸的质量体积百分比的含量≤6％，优选1-4％，更优选2％。
较佳地，所述滴剂优选滴眼液。
较佳地，所述滴剂为抑制眼部视网膜渗漏和/或降低视网膜新生血管数的滴剂，优选为治疗糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的滴剂。
较佳地，所述滴剂的每日滴加次数为1-3次，每次滴加量优选为0.3-0.8nmol蛋白/眼。
较佳地，所述滴剂以每日2次、每次1滴、持续3个月进行施用；和/或，所述滴剂以每日1次、每次1滴、持续5天进行施用；和/或，所述滴剂以每日2次、每次1滴、持续2周进行施用。
较佳地，编码所述转甲状腺素蛋白的核苷酸序列如SEQ ID NO:2所示。
较佳地，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架质粒中的启动子为鼠李唐诱导型启动子，优选为rhaPBAD启动子。
较佳地，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架载体为pET-21a或与其具有25％及以上同源性的载体，所述与其具有25％及以上同源性的载体的序列优选如SEQ ID NO:8所示。
较佳地，表达所述转甲状腺素蛋白的重组质粒的核苷酸序列如SEQ ID NO:3所示。
较佳地，所述转甲状腺素蛋白可以是在微生物细胞中进行表达(形成转化体的形式)，并进一步可以经过纯化；所述的微生物细胞可以为大肠杆菌，所述大肠杆菌包括但不限于E.coli BL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α、E.coli K12或E.coli TOP10。所述纯化可以是通过内毒素吸附柱(例如使用Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，通过0.22μm孔径滤膜去除残留菌体。
较佳地，表达所述转甲状腺素蛋白时，通过培养包含所述转甲状腺素蛋白的基因的转化体至所得菌体的OD 600达到1.5-2.0(例如1.6、1.7、1.8或1.9)时进行表达。
较佳地，表达所述转甲状腺素蛋白时为使用诱导表达的试剂进行诱导表达，所述诱导表达的试剂的质量体积百分比为0.1-2％，例如0.2％、0.3％、0.4％、0.5％、0.7％、0.8％、1.2％或1.6％，所述诱导表达的时间优选为8-20h，例如10h、12h、14h、16h、17h、18h或19h；所述诱导表达的试剂优选为鼠李糖或者IPTG。
较佳地，所述融合蛋白的序列如SEQ ID NO:6或SEQ ID NO:7所示。
较佳地，所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合表达；所述融合优选是将所述蛋白质类和/或多肽类药物融合在所述转甲状腺素蛋白的N端或C端；所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合优选是在微生物细胞中表达，并经过纯化。所述的微生物细胞可以为大肠杆菌，所述大肠杆菌包括但不限于E.coli BL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α、E.coli K12或E.coli TOP10。所述纯化优选是通过内毒素吸附柱去除内毒素，再通过0.22μm孔径滤膜去除残留菌体。
较佳地，所述蛋白质类和/或多肽类药物包括但不限于：溶菌酶、白蛋白和/或EGFR抗体，且分子量不超过45kDa；所述溶菌酶优选为鸡蛋清溶菌酶，其GenBank登录号为AAL69327.1；所述白蛋白优选为鸡蛋清白蛋白。
较佳地，所述蛋白质类和/或多肽类药物包括治疗眼部视网膜渗漏和/或视网膜血管新生类眼部疾病例如糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的蛋白质类和/或多肽类药物。
本发明人在实验过程中还意外发现，使用羧甲基纤维素或其盐例如羧甲基纤维素钠、硫酸软骨素或其盐例如硫酸软骨素A钠盐、右旋糖酐例如右旋糖酐70、透明质酸等材料能够与转甲状腺素蛋白和/或融合蛋白相互牵制、协同配合，形成一个有机整体，从而可以对与眼部血管新生和/或眼部视网膜渗漏等相关的眼部疾病例如糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变进行协同治疗，最终获得了较佳的治疗效果。而本领域技术人员仅知晓透明质酸、羧甲基纤维素或其盐例如羧甲基纤维素钠、硫酸软骨素或其盐例如硫酸软骨素A钠盐、右旋糖酐例如右旋糖酐70、透明质酸等材料具有湿润等作用，通常可以将它们用于制备滴眼液以能够有效缓解眼部干燥等。
基于此，上述滴剂中进一步可以含有药学上可接受的辅料，所述药学上可接受的辅料选自“羧甲基纤维素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”、“硫酸软骨素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”、“右旋糖酐或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”、和、“透明质酸或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”中的一种或多种。
较佳地，所述羧甲基纤维素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的黏度为800-1200CP。
较佳地，所述硫酸软骨素为硫酸软骨素A。
较佳地，所述右旋糖酐为右旋糖酐70。所述的右旋糖酐70的分子量通常在64000-76000范围内。
较佳地，所述透明质酸的分子量为10000-500000。
本发明中，所述的“药学上可接受的”通常是指无毒、安全，并且适合于患者使用。所述的“患者”优选哺乳动物，更优选为人类。
本发明中，所述的“或其盐”中的“盐”通常是药学上可接受的盐，其通常是指本发明化合物与相对无毒的、药学上可接受的酸或碱制备得到的盐。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的碱与这类化合物的原型接触的方式获得碱加成盐。药学上可接受的碱加成盐包括但不限于：锂盐、钠盐、钾盐、钙盐、铝盐、镁盐、锌盐、铋盐、铵盐、二乙醇胺盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的酸与这类化合物的原型接触的方式获得酸加成盐。所述的药学上可接受的酸包括无机酸，所述无机酸包括但不限于：盐酸、氢溴酸、氢碘酸、硝酸、碳酸、磷酸、亚磷酸、硫酸等。所述的药学上可接受的酸包括有机酸，所述有机酸包括但不限于：乙酸、丙酸、草酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、水杨酸、酒石酸、甲磺酸、异烟酸、酸式柠檬酸、油酸、单宁酸、泛酸、酒石酸氢、抗坏血酸、龙胆酸、富马酸、葡糖酸、糖酸、甲酸、乙磺酸、双羟萘酸(即4,4’-亚甲基-双(3-羟基-2-萘甲酸))、氨基酸(例如谷氨酸、精氨酸)等。当本发明的化合物中含有相对酸性和相对碱性的官能团时，可以被转换成碱加成盐或酸加成盐。具体可参见Berge et al.,"Pharmaceutical Salts",Journal of Pharmaceutical Science 66:1-19(1977)、或、Handbook of Pharmaceutical Salts:Properties,Selection,and Use(P.Heinrich Stahl and Camille G.Wermuth,ed.,Wiley-VCH,2002)。
在某一较佳实施例中，所述的“羧甲基纤维素或其盐”中所述的“盐”可为钠盐或钙盐，例如为羧甲基纤维素钠。
在某一较佳实施例中，所述的“硫酸软骨素或其盐”中所述的“盐”可为钠盐或钙盐，例如为硫酸软骨素A钠盐。
本发明中，所述的“溶剂合物”是指本发明化合物与化学计量或非化学计量的溶剂结合形成的物质。溶剂合物中的溶剂分子可以有序或非有序排列的形式存在。所述的溶剂包括但不限于：水、甲醇、乙醇等。
本发明中，所述的“药学上可接受的盐的溶剂合物”中的“药学上可接受的盐”和 “溶剂合物”如上所述，是指本发明化合物1、与相对无毒的、药学上可接受的酸或碱制备得到的2、与化学计量或非化学计量的溶剂结合形成的物质。所述的“药学上可接受的盐的溶剂合物”包括但不限于本发明所述辅料的盐酸一水合物。
本发明中，所述的“辅料”、“药学上可接受的盐”、“溶剂合物”和“药学上可接受的盐的溶剂合物”，以及下述的“化合物”、“糖苷”，可以以晶型或无定型的形式存在。术语“晶型”是指其中的离子或分子是按照一种确定的方式在三维空间作严格周期性排列，并具有间隔一定距离周期重复出现规律；因上述周期性排列的不同，可存在多种晶型，也即多晶型现象。术语“无定型”是指其中的离子或分子呈现杂乱无章的分布状态，即离子、分子间不具有周期性排列规律。
较佳地，所述滴剂中，所述羧甲基纤维素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的浓度为0-8mg/mL但不为0(即0＜浓度≤8mg/mL)，优选为2、4、6或8mg/mL。
较佳地，所述滴剂中，所述硫酸软骨素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的浓度为0-40mg/mL但不为0(即0＜浓度≤40mg/mL)，优选为10、20、30或40mg/mL。
较佳地，所述滴剂中，所述右旋糖酐或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的浓度为0-0.8mg/mL但不为0(即0＜浓度≤0.8mg/mL)，优选为0.2、0.4、0.6或0.8mg/mL。
较佳地，所述滴剂中，所述透明质酸或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的质量体积百分比的含量≤6％，优选1-4％，更优选2％。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、6mg/mL羧甲基纤维素钠和生理盐水组成。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、0.4mg/mL右旋糖酐70和生理盐水组成。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、20mg/mL硫酸软骨素A钠盐和生理盐水组成。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、2％透明质酸和生理盐水组成。
当所述滴剂含有上述药学上可接受的辅料时，所述滴剂的制备方法包括将所述的药学上可接受的辅料与所述的转甲状腺素蛋白和/或融合蛋白混合即可。
本发明人在实验过程中还意外发现，将转甲状腺素蛋白和/或融合蛋白与特定化合物共同施用眼部时，所述的化合物能够与转甲状腺素蛋白和/或融合蛋白协同配合、共同作用，从而能够有效治疗或缓解与眼部视网膜血管新生和/或眼部视网膜渗漏等相关的眼部疾病，使得治疗效果更佳。
基于此，上述滴剂中进一步可以含有化合物、其药学上可接受的盐、其糖苷、其溶剂合物、其药学上可接受的盐的溶剂合物、或其晶型；所述化合物选自双氯芬酸、维生素A和木犀草素中的一种或多种。
其中，双氯芬酸及其钠盐：双氯芬酸及其钠盐属于非甾体抗炎药，有明显的镇痛，消炎及解热作用。双氯芬酸钠滴眼液，用于治疗葡萄膜炎、角膜炎、巩膜炎，抑制角膜新生血管的形成；治疗眼内手术后、激光滤帘成形术后或各种眼部损伤的炎症反应；抑制白内障手术中缩瞳反应；用于准分子激光角膜切削术后止痛及消炎；治疗春季结膜炎、季节过敏性结膜炎等过敏性眼病；预防和治疗白内障及人工晶体术后炎症及黄斑囊样水肿；以及青光眼滤过术后促进滤过泡形成等。双氯芬酸钠滴眼液针对眼表使用或眼部开放式创伤使用，目前的市售产品有迪非，含量为1mg/mL。双氯芬酸、双氯芬酸钠的结构式如下所示：
Figure PCTCN2020128588-appb-000001
双氯芬酸，
Figure PCTCN2020128588-appb-000002
双氯芬酸钠。
维生素A：维生素A包括视黄醇(A1)、3-脱氢视黄醇(A2)，是一类脂溶性维生素，对热、酸、碱稳定。维生素A有促进生长、繁殖，维持骨骼、上皮组织、视力和粘膜上皮正常分泌等多种生理功能，维生素A及其类似物有阻止癌前期病变的作用。维生素A缺乏时表现为生长迟缓、暗适应能力减退而形成夜盲症。维生素A滴眼液用于促进角膜细胞的生长以及干眼症的治疗。其中视黄醇(A1)、3-脱氢视黄醇(A2)的结构式如下所 示：
Figure PCTCN2020128588-appb-000003
木犀草素：是一种天然黄酮类化合物，存在于多种植物中。具有多种药理活性，如消炎、抗过敏、降尿酸、抗肿瘤、抗菌、抗病毒等，临床主要用于止咳、祛痰、消炎、降尿酸、治疗心血管疾病，以及治疗肌萎缩性脊髓侧索硬化症、SARS、肝炎等。木犀草素，多以糖苷的形式存在于多种植物中。木犀草素及其糖苷形式的结构式分别如下所示：
Figure PCTCN2020128588-appb-000004
木犀草素，
Figure PCTCN2020128588-appb-000005
木犀草苷。
本发明中，所述的盐通常是药学上可接受的盐，具体如上所述。在某一较佳实施例中，所述的药学上可接受的盐为钠盐，例如为双氯芬酸钠。
较佳地，所述的糖苷为木犀草苷。
较佳地，所述的维生素A为维生素A1和/或维生素A2。在本发明某一较佳实施例 中，所述的维生素A可以购自国药，国药编号为CATOCCHM700908，CAS：68-26-8。
较佳地，所述滴剂中，所述双氯芬酸(或例如其钠盐双氯芬酸钠)的含量为5-20μmol/L，例如为10μmol/L。
较佳地，所述滴剂中，所述维生素A的含量为2-10μmol/L，例如为5μmol/L。
较佳地，所述滴剂中，所述木犀草素(或例如其糖苷形式木犀草苷)的含量为2-10μmol/L，例如为5μmol/L。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、辅料、10μmol/L双氯芬酸钠和生理盐水组成，所述辅料为6mg/mL羧甲基纤维素钠、0.4mg/mL右旋糖酐70、20mg/mL硫酸软骨素A钠盐以及2％(质量体积百分比)透明质酸中的任意一种。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、辅料、5μmol/L维生素A、5％(v/v)吐温80和生理盐水组成，所述辅料为6mg/mL羧甲基纤维素钠、0.4mg/mL右旋糖酐70、20mg/mL硫酸软骨素A钠盐以及2％(质量体积百分比)透明质酸中的任意一种。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、辅料、5μmol/L木犀草苷和生理盐水组成，所述辅料为6mg/mL羧甲基纤维素钠、0.4mg/mL右旋糖酐70、20mg/mL硫酸软骨素A钠盐以及2％(质量体积百分比)透明质酸中的任意一种。
当所述滴剂含有上述化合物时，所述滴剂的制备方法包括将所述化合物与所述的转甲状腺素蛋白和/或融合蛋白混合即可。
为了解决上述技术问题，本发明第三方面提供了一种滴剂(例如以滴眼液的形式)，其含有转甲状腺素蛋白、和/或转甲状腺素蛋白与药物组成的融合蛋白；所述药物为蛋白质类和/或多肽类药物，所述转甲状腺素蛋白如(a)、(b)或(c)所示：
(a)由SEQ ID NO:1所示氨基酸组成的蛋白质；
(b)在(a)中的氨基酸序列上经过取代、缺失或添加一个或几个氨基酸的序列所示的具有抑制血管新生功能的由(a)衍生的蛋白质；
(c)在(a)或(b)中的氨基酸序列上经过亲水性修饰或疏水性修饰的序列所示的蛋白质。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过22个氨基酸的取代。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过25个氨基酸的取代。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列的T3、T5、I26、N27、H31、R34、A36、A37、D38、D39、T40、S50、E61、E63、V65、I68、K70、I73、A81、H90、E92、P102、R104、T123、K126和/或E127发生取代。更佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过T3G、T5A、I26V、N27D、H31K、R34K、A36T、D39G、T40S、S50A、E61D、E63K、V65T、I68V、K70R、I73L、H90Y、P102H、R104H、T123S、K126Q和E127N的取代。更佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过T3G、T5A、I26V、N27D、H31K、R34K、A36T、A37S、D38E、D39G、T40S、S50A、E61D、E63K、I68V、K70R、I73L、A81T、H90F、E92D、P102H、R104H、T123S、K126Q和E127N的取代。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过5个氨基酸的缺失。更佳地，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列的第123-127位发生缺失。
较佳地，所述(b)中，所述的由(a)衍生的蛋白质的氨基酸序列优选如SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11所示。
较佳地，所述(c)中，所述亲水性修饰或疏水性修饰在(a)中的氨基酸序列上的第10位的半胱氨酸上进行。较佳地，所述疏水性修饰为使用长链疏水片段例如正十二烷进行修饰。在本发明某一较佳实施例中，所述蛋白质为在(a)中的氨基酸序列上的第10位的半胱氨酸通过马来酰亚胺连接正十二烷修饰的序列所示的蛋白质。本发明中，所述的在(a)中的氨基酸序列上的第10位的半胱氨酸还可以是连接有5-氨基荧光素等荧光标记，以便追踪修饰后的蛋白。
较佳地，当所述滴剂含有转甲状腺素蛋白与蛋白质类和/或多肽类药物组成的融合蛋白时，所述融合蛋白的含量4-30μmol/L，优选10-15μmol/L。
较佳地，当所述滴剂含有转甲状腺素蛋白时，所述转甲状腺素蛋白的含量为4-30μmol/L，优选5-30μmol/L，更优选10-20μmol/L，例如10、15、20μmol/L。
较佳地，所述滴剂中还含有生理盐水。本发明中，所述的生理盐水的使用量可按照本领域规定的标准进行使用即可。
较佳地，所述滴剂中还含有表面活性剂，所述表面活性剂例如为吐温80，其含量优选为5％(v/v)。
较佳地，所述滴剂中还含有透明质酸，所述透明质酸的质量体积百分比的含量≤6％，优选1-4％，更优选2％。
在本发明某一较佳实施例中，所述滴剂(例如以滴眼液的形式)还含有生理盐水与 1-6％透明质酸。
在本发明某一较佳实施例中，所述滴剂(例如以滴眼液的形式)还含有生理盐水与1-4％透明质酸。
在本发明某一较佳实施例中，所述滴剂(例如以滴眼液的形式)还含有生理盐水与2％透明质酸。
较佳地，所述滴剂优选滴眼液。
较佳地，所述滴剂为抑制眼部视网膜渗漏和/或降低视网膜新生血管数的滴剂，优选为治疗糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的滴剂。
较佳地，所述滴剂的每日滴加次数为1-3次，每次滴加量优选为0.3-0.8nmol蛋白/眼。
较佳地，所述滴剂以每日2次、每次1滴、持续3个月进行施用；和/或，所述滴剂以每日1次、每次1滴、持续5天进行施用；和/或，所述滴剂以每日2次、每次1滴、持续2周进行施用。
较佳地，编码所述转甲状腺素蛋白的核苷酸序列如SEQ ID NO:2所示。
较佳地，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架质粒中的启动子为鼠李唐诱导型启动子，优选为rhaPBAD启动子。
较佳地，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架载体为pET-21a或与其具有25％及以上同源性的载体，所述与其具有25％及以上同源性的载体的序列优选如SEQ ID NO:8所示。
较佳地，表达所述转甲状腺素蛋白的重组质粒的核苷酸序列如SEQ ID NO:3所示。
较佳地，所述转甲状腺素蛋白可以是在微生物细胞中进行表达(形成转化体的形式)，并进一步可以经过纯化；所述的微生物细胞可以为大肠杆菌，所述大肠杆菌包括但不限于E.coli BL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α、E.coli K12或E.coli TOP10。所述纯化可以是通过内毒素吸附柱(例如使用Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，通过0.22μm孔径滤膜去除残留菌体。
较佳地，表达所述转甲状腺素蛋白时，通过培养包含所述转甲状腺素蛋白的基因的转化体至所得菌体的OD 600达到1.5-2.0(例如1.6、1.7、1.8或1.9)时进行表达。
较佳地，表达所述转甲状腺素蛋白时为使用诱导表达的试剂进行诱导表达，所述诱导表达的试剂的质量体积百分比为0.1-2％，例如0.2％、0.3％、0.4％、0.5％、0.7％、0.8％、1.2％或1.6％，所述诱导表达的时间优选为8-20h，例如10h、12h、14h、16h、17h、 18h或19h；所述诱导表达的试剂优选为鼠李糖或者IPTG。
较佳地，所述融合蛋白的序列如SEQ ID NO:6或SEQ ID NO:7所示。
较佳地，所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合表达；所述融合优选是将所述蛋白质类和/或多肽类药物融合在所述转甲状腺素蛋白的N端或C端；所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合优选是在微生物细胞中表达，并经过纯化。所述的微生物细胞可以为大肠杆菌，所述大肠杆菌包括但不限于E.coli BL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α、E.coli K12或E.coli TOP10。所述纯化优选是通过内毒素吸附柱去除内毒素，再通过0.22μm孔径滤膜去除残留菌体。
较佳地，所述蛋白质类和/或多肽类药物包括但不限于：溶菌酶、白蛋白和/或EGFR抗体，且分子量不超过45kDa；所述溶菌酶优选为鸡蛋清溶菌酶，其GenBank登录号为AAL69327.1；所述白蛋白优选为鸡蛋清白蛋白。
较佳地，所述蛋白质类和/或多肽类药物包括治疗眼部视网膜渗漏和/或视网膜血管新生类眼部疾病例如糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的蛋白质类和/或多肽类药物。
在本发明某一较佳实施例中，上述滴剂例如滴眼液中转甲状腺素蛋白的含量为5-20μmol/L，每日2次，每次1滴，持续3个月。
在本发明某一较佳实施例中，上述滴剂例如滴眼液中转甲状腺素蛋白的含量为10-15μmol/L，每日2次。
在本发明某一较佳实施例中，上述滴剂例如滴眼液中转甲状腺素蛋白的含量为10μmol/L，每日2次。
在本发明某一较佳实施例中，上述滴剂例如滴眼液中转甲状腺素蛋白的含量为5-20μmol/L，每日1次，每次1滴，持续5天。
在本发明某一较佳实施例中，上述滴剂例如滴眼液中转甲状腺素蛋白的含量为10-15μmol/L，每日1次。
在本发明某一较佳实施例中，上述滴剂例如滴眼液中转甲状腺素蛋白的含量为10μmol/L，每日1次。
在本发明某一较佳实施例中，上述滴剂例如滴眼液中转甲状腺素蛋白的含量为5-20μmol/L，每日2次，每次1滴，持续2周。
在本发明某一较佳实施例中，上述滴剂例如滴眼液中转甲状腺素蛋白的含量为10-15μmol/L，每日2次。
在本发明某一较佳实施例中，上述滴剂例如滴眼液中转甲状腺素蛋白的含量为10 μmol/L，每日2次。
本发明中，所列的滴眼剂量和滴剂中转甲状腺素蛋白的含量仅为建议。本领域技术人员应当理解的是，根据本发明的剂量和含量进行适度调整以施用于合适的受试者时的用量也应该在本发明的保护范围内。
本发明人在实验过程中还意外发现，使用羧甲基纤维素或其盐例如羧甲基纤维素钠、硫酸软骨素或其盐例如硫酸软骨素A钠盐、右旋糖酐例如右旋糖酐70、透明质酸等材料能够与转甲状腺素蛋白和/或融合蛋白相互牵制、协同配合，形成一个有机整体，从而可以对与眼部血管新生和/或眼部视网膜渗漏等相关的眼部疾病例如糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变进行协同治疗，最终获得了较佳的治疗效果。而本领域技术人员仅知晓羧甲基纤维素或其盐例如羧甲基纤维素钠、硫酸软骨素或其盐例如硫酸软骨素A钠盐、右旋糖酐例如右旋糖酐70、透明质酸等材料具有湿润等作用，通常可以将它们用于制备滴眼液以能够有效缓解眼部干燥等。
基于此，上述滴剂中进一步可以含有药学上可接受的辅料，所述药学上可接受的辅料选自“羧甲基纤维素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”、“硫酸软骨素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”、“右旋糖酐或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”、和“透明质酸或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”中的一种或多种。
较佳地，所述羧甲基纤维素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的黏度为800-1200CP。
较佳地，所述硫酸软骨素为硫酸软骨素A。
较佳地，所述右旋糖酐为右旋糖酐70。所述的右旋糖酐70的分子量通常在64000-76000范围内。
较佳地，所述透明质酸的分子量为10000-500000。
在某一较佳实施例中，所述的“羧甲基纤维素或其盐”中所述的“盐”可为钠盐或钙盐，例如为羧甲基纤维素钠。
在某一较佳实施例中，所述的“硫酸软骨素或其盐”中所述的“盐”可为钠盐或钙盐，例如为硫酸软骨素A钠盐。
较佳地，所述滴剂中，所述羧甲基纤维素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的浓度为0-8mg/mL但不为0(即0＜浓度≤8mg/mL)，优选为 2、4、6或8mg/mL。
较佳地，所述滴剂中，所述硫酸软骨素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的浓度为0-40mg/mL但不为0(即0＜浓度≤40mg/mL)，优选为10、20、30或40mg/mL。
较佳地，所述滴剂中，所述右旋糖酐或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的浓度为0-0.8mg/mL但不为0(即0＜浓度≤0.8mg/mL)，优选为0.2、0.4、0.6或0.8mg/mL。
较佳地，所述滴剂中，所述透明质酸或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的质量体积百分比的含量≤6％，优选1-4％，更优选2％。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、6mg/mL羧甲基纤维素钠和生理盐水组成。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、0.4mg/mL右旋糖酐70和生理盐水组成。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、20mg/mL硫酸软骨素A钠盐和生理盐水组成。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、2％透明质酸和生理盐水组成。
当所述滴剂含有上述药学上可接受的辅料时，所述滴剂的制备方法包括将所述的药学上可接受的辅料与所述的转甲状腺素蛋白和/或融合蛋白混合即可。
本发明人在实验过程中还意外发现，将转甲状腺素蛋白和/或融合蛋白与特定化合物共同施用眼部时，所述的化合物能够与转甲状腺素蛋白和/或融合蛋白协同配合、共同作用，从而能够有效治疗或缓解与眼部视网膜血管新生和/或眼部视网膜渗漏等相关的眼部疾病，使得治疗效果更佳。
基于此，上述滴剂中进一步可以含有化合物、其药学上可接受的盐、其糖苷、其溶剂合物、其药学上可接受的盐的溶剂合物、或其晶型；所述化合物选自双氯芬酸、维生素A和木犀草素中的一种或多种。
本发明中，所述的盐通常是药学上可接受的盐，具体如上所述。在某一较佳实施例中，所述的药学上可接受的盐为钠盐，例如为双氯芬酸钠。
较佳地，所述的糖苷为木犀草苷。
较佳地，所述的维生素A为维生素A1和/或维生素A2。在本发明某一较佳实施例 中，所述的维生素A可以购自国药，国药编号为CATOCCHM700908，CAS：68-26-8。
较佳地，所述滴剂中，所述双氯芬酸(或例如其钠盐双氯芬酸钠)的含量为5-20μmol/L，例如为10μmol/L。
较佳地，所述滴剂中，所述维生素A的含量为2-10μmol/L，例如为5μmol/L。
较佳地，所述滴剂中，所述木犀草素(或例如其糖苷形式木犀草苷)的含量为2-10μmol/L，例如为5μmol/L。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、辅料、10μmol/L双氯芬酸钠和生理盐水组成，所述辅料为6mg/mL羧甲基纤维素钠、0.4mg/mL右旋糖酐70、20mg/mL硫酸软骨素A钠盐以及2％(质量体积百分比)透明质酸中的任意一种。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、辅料、5μmol/L维生素A、5％(v/v)吐温80和生理盐水组成，所述辅料为6mg/mL羧甲基纤维素钠、0.4mg/mL右旋糖酐70、20mg/mL硫酸软骨素A钠盐以及2％(质量体积百分比)透明质酸中的任意一种。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、辅料、5μmol/L木犀草苷和生理盐水组成，所述辅料为6mg/mL羧甲基纤维素钠、0.4mg/mL右旋糖酐70、20mg/mL硫酸软骨素A钠盐以及2％(质量体积百分比)透明质酸中的任意一种。
当所述滴剂含有上述化合物时，所述滴剂的制备方法包括将所述化合物与所述的转甲状腺素蛋白和/或融合蛋白混合即可。
本发明中，所述的转甲状腺素蛋白是一种四聚体载体蛋白，可在血浆和脑脊液中运输甲状腺激素。研究发现转甲状腺素蛋白进入细胞是通过高密度脂蛋白受体SRB1所介导的(Landers,K.A.,et al.,Transthyretin uptake in placental cells is regulated by the high-density lipoprotein receptor,scavenger receptor class B member 1.Mol Cell Endocrinol,2018.474:p.89-96)。从结构上看(三维结构见图1)，转甲状腺素蛋白的四聚体表面具有显著的亲水结构域(深色)与疏水结构域(浅色)，转甲状腺素蛋白的核心疏水结构域能够携带强疏水的甲状腺素分子跨越各种细胞(见图2)。此外，不同物种的转甲状腺素蛋白的氨基酸序列高度保守，人源的转甲状腺素蛋白与SD大鼠及C57BL/6小鼠来源的转甲状腺素蛋白的氨基酸序列相似度>95％(见图3)。
本发明人在制备滴剂的过程中，发现转甲状腺素蛋白在大肠杆菌内的重组表达水平极低，大部分为不可溶的包涵体形式。常用的提高蛋白表达量的方法有表达载体选择，发酵条件优化(温度、pH值、时间、诱导剂浓度等)或者分子伴侣共表达辅助等，但是本发明人尝试了上述知悉方法均不能有效提升转甲状腺素蛋白在大肠杆菌中的表达水平。 本发明人经过大量实验，发现基于pET-21a质粒、利用鼠李唐诱导型(例如rhaPBAD启动子)替代质粒上的启动子(例如T7启动子)后，或对TTR的核苷酸序列进行密码子优化(核苷酸序列如SEQ ID NO:2所示)后，或对于pET-21a质粒本身进行序列重构后，或对表达蛋白时的OD值、诱导蛋白表达的试剂的用量、诱导表达的时间进行优化后，能够构建转甲状腺素蛋白成熟片段(NCBI Reference Sequence:NP_000362.1)的高效生产质粒，从而使得最终所得转甲状腺素蛋白的表达量显著提升。因此，本发明中进一步提供了一种表达转甲状腺素蛋白的基因、包含其的重组表达载体和转化体，一种用于表达转甲状腺素蛋白的重组质粒，一种转甲状腺素蛋白的表达方法等。使用本发明的表达转甲状腺素蛋白的基因、或使用表达转甲状腺素蛋白的重组质粒进行表达、或使用本发明所述的转甲状腺素蛋白的表达方法时，转甲状腺素蛋白的表达量显著提高。具体的：
本发明第四方面还提供了一种表达转甲状腺素蛋白的基因，所述基因的核苷酸序列如SEQ ID NO:2所示。
本发明第五方面还提供了一种重组表达载体，所述重组表达载体中含如本发明第四方面所述的基因。
较佳地，所述重组表达载体的骨架载体中的启动子为鼠李唐诱导型启动子；所述鼠李唐诱导型启动子优选为rhaPBAD启动子。
较佳地，所述重组表达载体的骨架载体为pET-21a或与pET-21a具有25％及以上同源性的载体，所述与pET-21a具有25％及以上同源性的载体的序列优选如SEQ ID NO:8所示。
更佳地，所述重组表达载体的核苷酸序列如SEQ ID NO:3所示。
本发明第六方面还提供了一种重组质粒，其序列如SEQ ID NO:8所示。
本发明第七方面还提供了一种表达转甲状腺素蛋白的重组质粒，所述重组质粒包含骨架质粒和转甲状腺素蛋白的表达片段，
其中，所述骨架质粒中的启动子为鼠李唐诱导型启动子；和/或，所述骨架质粒的骨架载体为pET-21a或与其具有25％及以上同源性的载体，所述与其具有25％及以上同源性的载体的序列优选如SEQ ID NO:8所示。
较佳地，编码所述转甲状腺素蛋白的表达片段的核苷酸序列如SEQ ID NO:2所示。
较佳地，所述鼠李唐诱导型启动子为rhaPBAD启动子。
更佳地，所述重组质粒的核苷酸序列如SEQ ID NO:3所示。
本发明第八方面还提供了一种转化体，其包括如本发明第四方面所述的基因、或者如本发明第五方面所述的重组表达载体、或者如本发明第六或七方面所述的重组质粒。
较佳地，在宿主中导入如本发明第四方面所述的基因、如本发明第五方面所述的重组表达载体或者如本发明第六或七方面所述的重组质粒，所述宿主为大肠杆菌，优选为大肠杆菌E.coli BL21(DE3)、E.coli TG1、E.coli JM109、E.coli DH5α、E.coli K12或E.coli TOP10。
本发明第九方面还提供了一种转甲状腺素蛋白的制备方法，其包括以下步骤：
(1)获得如本发明第八方面所述的转化体；
(2)筛选所述转化体，表达并纯化所述蛋白。
较佳地，所述表达为培养所述转化体至所得菌体的OD 600达到1.5-2.0(例如1.6、1.7、1.8或1.9)时进行表达。
较佳地，所述表达为使用诱导表达的试剂进行诱导表达，所述诱导表达的试剂的质量体积百分比为0.1-2％，例如0.2％、0.3％、0.4％、0.5％、0.7％、0.8％、1.2％或1.6％，所述诱导表达的时间优选为8-20h，例如10h、12h、14h、16h、17h、18h或19h；所述诱导表达的试剂优选为鼠李糖或者IPTG。
本发明第十方面还提供了一种转甲状腺素蛋白的表达方法，其包括获得包含转甲状腺素蛋白的基因的转化体并筛选、表达、纯化所述转甲状腺素蛋白的步骤。
较佳地，所述表达为使用诱导表达的试剂进行诱导表达，所述诱导表达的试剂的质量体积百分比为0.1-2％，例如0.2％、0.3％、0.4％、0.5％、0.7％、0.8％、1.2％或1.6％，所述诱导表达的时间优选为8-20h，例如10h、12h、14h、16h、17h、18h或19h；所述诱导表达的试剂优选为鼠李糖或者IPTG。
基于上述所述，本发明人通过大量实验，还意外地发现在转甲状腺素蛋白跨越角膜屏障进入玻璃体及眼底之后，转甲状腺素蛋白可以显著抑制眼球视网膜渗漏，显著降低视网膜新生血管数，有效缓解DR、AMD、ROP等眼部疾病。因此，本发明中还进一步提供了转甲状腺素蛋白在制备抑制眼部视网膜渗漏和/或降低视网膜新生血管数的药物中的应用，例如，在制备治疗糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病等眼部疾病的药物中的应用。
本发明第十一方面还提供了转甲状腺素蛋白和/或如本发明第三方面所述的滴剂在制备抑制眼部视网膜渗漏和/或降低视网膜新生血管数的药物中的应用，优选为在制备治疗老年性黄斑变性、早产儿视网膜病变和/或糖尿病视网膜病变的药物中的应用。
较佳地，所述转甲状腺素蛋白以非注射剂型的方式存在，所述非注射剂型优选抹剂或滴剂，所述抹剂优选膏体(如眼药膏)或凝胶(如眼用凝胶)，所述滴剂例如为滴眼液 (所述滴剂中的组分、使用剂量等可以如上所述)。
较佳地，使用如本发明第四方面所述的基因、如本发明第五方面所述的重组表达载体或者如本发明第六或七方面所述的重组质粒、如本发明第八方面所述的转化体表达所述的转甲状腺素蛋白。
本发明第十二方面还提供了转甲状腺素蛋白和/或如本发明第三方面所述的滴剂在治疗眼部疾病中的应用。所述的眼部疾病优选为眼部视网膜渗漏和/或视网膜血管新生相关眼部疾病，更优选为糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变。所述的转甲状腺素蛋白优选如上所述。所述应用中使用的条件、剂量、施用方法等均可以如上所述进行应用。
本发明第十三方面还提供了一种治疗眼部视网膜渗漏和/或视网膜血管新生类眼部疾病例如糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变等的方法，其包括施用上述转甲状腺素蛋白和/或如本发明第三方面所述的滴剂。所述方法中治疗的条件、剂量、施用方法等均可以如上所述进行。
本发明第十四方面还提供了一种使用上述的转甲状腺素蛋白将外源蛋白和/或多肽通过眼部屏障输送至眼内的方法。
在本发明的某一较佳实施例中，上述融合蛋白的制备方法为：(1)将转甲状腺素蛋白与药物蛋白和/或多肽的编码基因连接在pET 21a(+)质粒上，获得重组质粒；(2)将步骤(1)构建的重组质粒转化至宿主细胞内进行表达；(3)使用TB培养基，发酵温度为30-40℃，OD 600达到1.5-2.0时利用0.1-0.5mM IPTG进行诱导，诱导时间为8-16h；(4)通过镍柱亲和吸附纯化制备表达的目标蛋白后，通过内毒素吸附柱(Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，通过0.22μm孔径滤膜去除残留菌体。
在本发明的某一较佳实施例中，所述转甲状腺素蛋白的表达和纯化的步骤包括：将构建的pETx-rhaPBAD-ttr质粒(质粒整体的核酸序列如SEQ ID NO:3所示)转化入E.coli BL21(DE3)细胞，将所得重组E.coli BL21(DE3)在LB培养基中培养，制备种子液，再以5％的接种量，接入至5L TB培养基中，温度37℃，搅拌桨转速150rpm，培养至OD 600为1.5-2.0；加入0.4-2％鼠李糖诱导16-20h。使用内毒素吸附柱(Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，通过0.22μm孔径滤膜去除残留菌体。
基于上述所述本发明人在实验过程中还意外发现，使用羧甲基纤维素或其盐例如羧 甲基纤维素钠、硫酸软骨素或其盐例如硫酸软骨素A钠盐、右旋糖酐例如右旋糖酐70、透明质酸等材料能够与转甲状腺素蛋白和/或融合蛋白相互牵制、协同配合，形成一个有机整体，从而可以对与眼部血管新生和/或眼部视网膜渗漏等相关的眼部疾病例如糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变进行协同治疗，最终获得了较佳的治疗效果。
本发明第十五方面还提供了一种眼用制剂，其包括转甲状腺素蛋白和药学上可接受的辅料，所述药学上可接受的辅料选自“羧甲基纤维素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”、“硫酸软骨素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”、“右旋糖酐或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”、和、“透明质酸或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型”中的一种或多种。
较佳地，所述羧甲基纤维素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的黏度为800-1200CP。
较佳地，所述硫酸软骨素为硫酸软骨素A。
较佳地，所述右旋糖酐为右旋糖酐70。所述的右旋糖酐70的分子量通常在64000-76000范围内。
较佳地，所述透明质酸的分子量为10000-500000。
在某一较佳实施例中，所述的“羧甲基纤维素或其盐”中所述的“盐”可为钠盐或钙盐，例如为羧甲基纤维素钠。
在某一较佳实施例中，所述的“硫酸软骨素或其盐”中所述的“盐”可为钠盐或钙盐，例如为硫酸软骨素A钠盐。
较佳地，所述眼用制剂中，所述羧甲基纤维素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的浓度为0-8mg/mL但不为0(即0＜浓度≤8mg/mL)，优选为2、4、6或8mg/mL。
较佳地，所述眼用制剂中，所述硫酸软骨素或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的浓度为0-40mg/mL但不为0(即0＜浓度≤40mg/mL)，优选为10、20、30或40mg/mL。
较佳地，所述眼用制剂中，所述右旋糖酐或其盐、其溶剂合物、其药学上可接受的盐的溶剂合物、或、其晶型的浓度为0-0.8mg/mL但不为0(即0＜浓度≤0.8mg/mL)，优选为0.2、0.4、0.6或0.8mg/mL。
较佳地，所述滴剂中，所述透明质酸或其盐、其溶剂合物、其药学上可接受的盐的溶 剂合物、或、其晶型的质量体积百分比的含量≤6％，优选1-4％，更优选2％。
较佳地，所述眼用制剂中，所述转甲状腺素蛋白如(a)、(b)或(c)所示：
(a)由SEQ ID NO.1所示氨基酸组成的蛋白质；
(b)在(a)中的氨基酸序列上经过取代、缺失或添加一个或几个氨基酸的序列所示具有抑制血管新生功能的由(a)衍生的蛋白质；
(c)在(a)或(b)中的氨基酸序列上经过亲水性修饰或疏水性修饰的序列所示的蛋白质。
其中，所述(b)中，所述的由(a)衍生的蛋白质的氨基酸序列可以是如SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11所示。
其中，所述(c)中，所述亲水性修饰或疏水性修饰可以是在(a)中的氨基酸序列上的第10位的半胱氨酸上进行，优选在(a)中的氨基酸序列上的第10位的半胱氨酸上使用长链疏水片段例如正十二烷进行所述修饰、或、在(a)中的氨基酸序列上的第10位的半胱氨酸上通过使用马来酰亚胺连接正十二烷进行所述修饰。
较佳地，所述眼用制剂中含有的所述转甲状腺素蛋白的含量为4-30μmol/L，优选5-30μmol/L，更优选10-20μmol/L，例如10、15或20μmol/L。
较佳地，所述眼用制剂中还可含有本领域常规使用的其他药学上可接受的辅料，例如生理盐水等。本发明中，所述的生理盐水的使用量可按照本领域规定的标准进行使用即可。
较佳地，所述眼用制剂可以是以本领域常规施用于眼部的产品形式存在，例如可以为滴剂例如滴眼液，还可以为喷剂、凝胶剂或眼用脂质体等。
较佳地，所述眼用制剂为抑制眼部视网膜渗漏和/或降低视网膜新生血管数的眼用制剂；所述眼用制剂优选为治疗糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的眼用制剂。
较佳地，所述眼用制剂以每日施用1-3次、每次施用量优选为0.3-0.8nmol蛋白/眼进行施用。
较佳地，所述眼用制剂以每日2次、每次1滴、持续3个月进行施用。
较佳地，所述眼用制剂以每日1次、每次1滴、持续5天进行施用。
较佳地，所述眼用制剂以每日2次、每次1滴、持续2周进行施用。
在某一较佳实施例中，所述眼用制剂由10μmol/L转甲状腺素蛋白、6mg/mL羧甲基纤维素钠和生理盐水组成。
在某一较佳实施例中，所述眼用制剂由10μmol/L转甲状腺素蛋白、0.4mg/mL右旋 糖酐70和生理盐水组成。
在某一较佳实施例中，所述眼用制剂由10μmol/L转甲状腺素蛋白、20mg/mL硫酸软骨素A钠盐和生理盐水组成。
在某一较佳实施例中，所述滴剂由10μmol/L转甲状腺素蛋白、2％透明质酸和生理盐水组成。
基于上述所述，本发明人在实验过程中还意外发现，将转甲状腺素蛋白和/或融合蛋白与特定化合物共同施用眼部时，所述的化合物能够与转甲状腺素蛋白和/或融合蛋白协同配合、共同作用，从而能够有效治疗或缓解与眼部视网膜血管新生和/或眼部视网膜渗漏等相关的眼部疾病，使得治疗效果更佳。
本发明第十六方面还提供了一种眼用制剂，其包括化合物、其药学上可接受的盐、其糖苷、其溶剂合物、其药学上可接受的盐的溶剂合物、或其晶型，以及转甲状腺素蛋白；所述化合物选自双氯芬酸、维生素A和木犀草素中的一种或多种。
在某一较佳实施例中，所述的药学上可接受的盐为钠盐，例如为双氯芬酸钠。
较佳地，所述的糖苷为木犀草苷。
较佳地，所述的维生素A为维生素A1和/或维生素A2。在本发明某一较佳实施例中，所述的维生素A可以购自国药，国药编号为CATOCCHM700908，CAS：68-26-8。
较佳地，所述眼用制剂中，所述转甲状腺素蛋白的含量为4-30μmol/L，优选5-30μmol/L，更优选10-20μmol/L，例如为10、15或20μmol/L。
较佳地，所述眼用制剂中，所述双氯芬酸(或例如其钠盐双氯芬酸钠)的含量为5-20μmol/L，例如为10μmol/L。
较佳地，所述眼用制剂中，所述维生素A的含量为2-10μmol/L，例如为5μmol/L。
较佳地，所述眼用制剂中，所述木犀草素(或例如其糖苷形式木犀草苷)的含量为2-10μmol/L，例如为5μmol/L。
较佳地，所述眼用制剂中，所述转甲状腺素蛋白如(a)、(b)或(c)所示：
(a)由SEQ ID NO.1所示氨基酸组成的蛋白质；
(b)在(a)中的氨基酸序列上经过取代、缺失或添加一个或几个氨基酸的序列所示具有抑制血管新生功能的由(a)衍生的蛋白质；
(c)在(a)或(b)中的氨基酸序列上经过亲水性修饰或疏水性修饰的序列所示的蛋白质。
其中，所述(b)中，所述的由(a)衍生的蛋白质的氨基酸序列可以是如SEQ ID NO: 9、SEQ ID NO:10或SEQ ID NO:11所示。
其中，所述(c)中，所述亲水性修饰或疏水性修饰在(a)中的氨基酸序列上的第10位的半胱氨酸上进行，优选在(a)中的氨基酸序列上的第10位的半胱氨酸上使用长链疏水片段例如正十二烷进行所述修饰、或、在(a)中的氨基酸序列上的第10位的半胱氨酸上通过使用马来酰亚胺连接正十二烷进行所述修饰。
较佳地，所述眼用制剂中还含有药学上可接受的辅料例如生理盐水。本发明中，所述的生理盐水的使用量可按照本领域规定的标准进行使用即可。
较佳地，所述眼用制剂中还含有药学上可接受的辅料例如表面活性剂，所述表面活性剂例如为吐温80，其含量优选为5％(v/v)。
较佳地，所述眼用制剂中还含有药学上可接受的辅料，所述辅料为羧甲基纤维素钠、右旋糖酐70、硫酸软骨素A钠盐或透明质酸。所述羧甲基纤维素钠的浓度为0-8mg/mL但不为0(即0＜浓度≤8mg/mL)，优选为2、4、6或8mg/mL。所述右旋糖酐70的浓度为0-0.8mg/mL但不为0(即0＜浓度≤0.8mg/mL)，优选为0.2、0.4、0.6或0.8mg/mL。所述硫酸软骨素A钠盐的浓度为0-40mg/mL但不为0(即0＜浓度≤40mg/mL)，优选为10、20、30或40mg/mL。所述透明质酸的质量体积百分比的含量优选≤6％，优选1-4％，更优选2％。
较佳地，所述眼用制剂可以是以本领域常规施用于眼部的产品形式存在，例如可以为滴剂例如滴眼液，还可以为喷剂、凝胶剂或眼用脂质体等。
较佳地，所述眼用制剂以每日施用1-3次、每次施用量优选为0.3-0.8nmol蛋白/眼进行施用。
较佳地，所述眼用制剂以每日2次、每次1滴、持续3个月进行施用。
较佳地，所述眼用制剂以每日1次、每次1滴、持续5天进行施用。
较佳地，所述眼用制剂以每日2次、每次1滴、持续2周进行施用。
在某一较佳实施例中，所述眼用制剂由10μmol/L转甲状腺素蛋白、辅料、10μmol/L双氯芬酸钠和生理盐水组成，所述辅料为6mg/mL羧甲基纤维素钠、0.4mg/mL右旋糖酐70、20mg/mL硫酸软骨素A钠盐以及2％(质量体积百分比)透明质酸中的任意一种。
在某一较佳实施例中，所述眼用制剂由10μmol/L转甲状腺素蛋白、辅料、5μmol/L维生素A、5％(v/v)吐温80和生理盐水组成，所述辅料为6mg/mL羧甲基纤维素钠、0.4mg/mL右旋糖酐70、20mg/mL硫酸软骨素A钠盐以及2％(质量体积百分比)透明质酸中的任意一种。
在某一较佳实施例中，所述眼用制剂由10μmol/L转甲状腺素蛋白、辅料、5μmol/L 木犀草苷和生理盐水组成，所述辅料为6mg/mL羧甲基纤维素钠、0.4mg/mL右旋糖酐70、20mg/mL硫酸软骨素A钠盐以及2％(质量体积百分比)透明质酸中的任意一种。
较佳地，所述眼用制剂为抑制眼部视网膜渗漏和/或降低视网膜新生血管数的眼用制剂，所述眼用制剂优选为治疗糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的眼用制剂。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明中，所述的药学上可接受的辅料等通常可以通过商购获得，例如购自国药集团。
本发明中，所提及的化合物等通常可以通过商购获得，例如购自国药集团、百灵威科技有限公司等。
本发明中，所述的“包括或包含”可以是指除了包括后面所列举的成分，还存在其他成分；在某些情况下，也可以是指“由……组成”，即只包括后面所列举的成分而不存在其他成分。
本发明中的氨基酸简写符号如无特殊说明均为本领域常规，具体简写符号对应的氨基酸如表1-1所示。
表1-1
氨基酸名称 三字母符号 单字母符号 氨基酸名称 三字母符号 单字母符号
丙氨酸(alanine) Ala A 亮氨酸(leucine) Leu L
精氨酸(arginine) Arg R 赖氨酸(lysine) Lys K
天冬酰胺(asparagine) Asn N 甲硫氨酸(methionine) Met M
天冬氨酸(asparticacid) Asp D 苯丙氨酸(phenylalanine) Phe F
半胱氨酸(cysteine) Cys C 脯氨酸(proline) Pro P
谷氨酰胺(glutanine) Gln Q 丝胺酸(serine) Ser S
谷氨酸(glutamicacid) Glu E 苏氨酸(threonine) Thr T
甘氨酸(Glicine) Gly G 色氨酸(tryptophan) Trp W
组氨酸(histidine) His H 酪氨酸(tyrosine) Tyr Y
异亮氨酸(isoleucine) Ile I 颉氨酸(valine) Val V
所述氨基酸对应的密码子也为本领域常规，具体氨基酸与密码子的对应关系如表1-2所示。
表1-2
Figure PCTCN2020128588-appb-000006
本发明的积极进步效果在于：转甲状腺素蛋白在人体内具有较好的生物相容性与安全性，不仅自身能够通过眼部屏障，还能有效地将外源蛋白和/或多肽输送到眼内，达到治疗眼部疾病的作用，在医药领域作为注射药物的替代物方面具有重要的应用前景。本发明中，通过使用滴剂进行滴眼而非直接注射的方式，转甲状腺素蛋白在治疗DR、AMD、ROP等眼部疾病时，转甲状腺素蛋白可跨越角膜屏障进入玻璃体及眼底，显著抑制眼球 视网膜渗漏，显著降低视网膜新生血管数，有效缓解DR、AMD、ROP等眼部疾病的病理现象。
附图说明
图1显示了转甲状腺素蛋白(TTR)的三维结构(PDB ID：1ICT)。
图2显示了TTR核心疏水结构域携带强疏水的甲状腺素分子跨越各种细胞。
图3显示了人源TTR与SD大鼠、C57BL/6小鼠、家兔来源的TTR氨基酸序列相似度>95％，其中，SD大鼠来源的TTR氨基酸序列如SEQ ID NO:9所示，C57BL/6小鼠来源的TTR氨基酸序列如SEQ ID NO:10所示。
图4为pET 21a(+)-His-tag-TTR-X质粒图。基因序列前端融合表达His-tag序列，并通过Nde I与EcoR I两个限制性内切酶酶切位点连接入质粒；“X”表示与TTR融合的蛋白。
图5为E.coli BL21(DE3)表达人源转甲状腺素蛋白，融合蛋白纯化产物以及绿色荧光蛋白，鸡蛋清溶菌酶与鸡蛋清白蛋白标准品的电泳图谱。
图6显示了TTR滴眼后C57BL/6小鼠及SD大鼠的角膜、玻璃体与眼底样本(视网膜，脉络膜)中TTR的含量。
图7为人源转甲状腺素蛋白及其融合蛋白对大鼠及家兔滴眼两周后，玻璃体腔内目标蛋白的western-blot图谱，左眼为滴加眼，右眼为对照眼。由于人源/大鼠源/兔源TTR具有较高的同源性，TTR经过滴眼后，玻璃体样本使用抗TTR抗体检测存在本底TTR阳性信号；而使用抗His-tag抗体检测时，滴加眼内阳性信号显著上升，说明人源TTR可以有效进入眼内到达玻璃体；TTR与GFP，Lysozyme以及Ovalbumin等外源蛋白融合表达后能有效进入眼内，而未融合表达的上述蛋白则无法进入。
图8显示了TTR滴眼STZ诱导SD大鼠后的视网膜渗漏与视网膜新生血管数。
图9显示了TTR滴眼阻止了ROP的造模过程。
图10显示了TTR滴眼抑制了ROP的病理过程。
图11显示了TTR滴眼抑制了AMD模型的病理过程。
图12显示了化学修饰人源TTR的过程。
图13A显示了蛋白结构为TTR二聚体，双氯芬酸配体分子使用箭头指示，一分子TTR二聚体可以结合两分子双氯芬酸。
图13B显示了双氯芬酸与TTR氨基酸残基的相互作用。
图14A显示了通过Discovery studio软件进行分子模拟发现维生素A1能够与TTR多 聚体的疏水通道稳定结合，图中蛋白结构为TTR二聚体，维生素A1配体分子使用箭头指示，一分子TTR二聚体可以结合一分子维生素A1。
图14B显示了维生素A1与TTR氨基酸残基的相互作用。
图15A显示了通过Discovery studio软件进行分子模拟发现维生素A2能够与TTR多聚体的疏水通道稳定结合，图中蛋白结构为TTR二聚体，维生素A2配体分子使用箭头指示，一分子TTR二聚体可以结合一分子维生素A2。
图15B显示了维生素A2与TTR氨基酸残基的相互作用。
图16A显示了通过Discovery studio软件进行分子模拟发现木犀草苷能够与TTR多聚体稳定结合，图中蛋白结构为TTR二聚体，木犀草苷配体分子使用箭头指示，一分子TTR二聚体可以结合一分子木犀草苷。
图16B显示了木犀草苷与TTR氨基酸残基的相互作用。
图17A显示了通过Discovery studio软件进行分子模拟发现磺胺甲恶唑能够与TTR多聚体稳定结合，图中蛋白结构为TTR二聚体，磺胺甲恶唑配体分子使用箭头指示，一分子TTR二聚体可以结合一分子磺胺甲恶唑。
图17B显示了磺胺甲恶唑与TTR氨基酸残基的相互作用。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
第一部分：转甲状腺素蛋白(TTR)及其融合蛋白的制备
实施例1：转甲状腺素蛋白的制备
转甲状腺素蛋白的制备包括如下步骤：
(1)重组质粒pET 21a(+)-His-tag-TTR的构建：合成核苷酸序列如SEQ ID NO:4所示的His-tag-TTR(其中所用TTR的氨基酸序列如SEQ ID NO:1所示，pET 21a购于ATCC中国菌种保藏中心)，将其用Nde I和EcoR I酶与pET 21a(+)连接，经测序验证(测序公司为南京金斯瑞生物科技有限公司，下同)，构建成功。
(2)重组TTR的表达和纯化：将步骤(1)构建的pET 21a(+)-His-tag-TTR质粒转化入E.coli BL21(DE3)细胞，将所得重组E.coli BL21(DE3)在LB培养基中培养，制备种子液，再以5％的接种量，接入至5L TB培养基中，温度37℃，搅拌桨转速150rpm，培养至OD 600为1.5-2.0；加入0.1-0.5mM IPTG诱导8-16h(表1)。利用高压均质 破菌并将上清液通过镍柱亲和吸附制备得到TTR。所得蛋白通过内毒素吸附柱(Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，通过0.22μm孔径滤膜去除残留菌体。对TTR蛋白产量进行检测，结果如表1所示。在OD 600为1.5-1.8时，以0.3-0.5mM IPTG诱导12-14h，可获得的蛋白产量≥17mg/g湿菌体。
表1不同诱导条件下TTR的表达
Figure PCTCN2020128588-appb-000007
实施例2：转甲状腺素蛋白-绿色荧光蛋白的融合蛋白的制备
转甲状腺素蛋白-绿色荧光蛋白的融合蛋白(TTR-GFP)按照如下方法制备：
(1)重组质粒pET 21a(+)-His-tag-TTR-GFP(以下重组质粒pET 21a(+)-His-tag-TTR-X质粒图如图4所示)的构建：合成如SEQ ID NO:5所示的His-tag-TTR-GFP序列，将其用Nde I和EcoR I酶与pET 21a(+)连接，经测序验证，构建成功。
(2)TTR-GFP融合蛋白的表达和纯化：将步骤(1)构建的重组质粒转化入E.coli BL21(DE3)细胞，将所得重组E.coli BL21(DE3)在LB培养基中培养，制备种子液，以5％的接种量，接入5L TB培养基中，温度37℃，搅拌桨转速150rpm，培养OD 600至1.5-2.0；加入0.1-0.5mM IPTG进行诱导8-16h；利用高压均质破菌并将上清液通过镍柱亲和吸附制备得到TTR-GFP融合蛋白。所得蛋白通过内毒素吸附柱(Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，通过0.22μm孔径滤膜去除残留菌体。对TTR-GFP蛋白产量进行检测，结果如表2所示。在OD 600为1.5-1.9时，以0.3-0.5mM IPTG诱导12h，可获得的蛋白产量≥10mg/g湿菌体。
表2不同诱导条件下TTR-GFP的表达
Figure PCTCN2020128588-appb-000008
实施例3：转甲状腺素蛋白-鸡蛋清溶酶菌的融合蛋白的制备
转甲状腺素蛋白-鸡蛋清溶酶菌的融合蛋白(TTR-Lysozyme)按照如下方法制备：
(1)重组质粒pET 21a(+)-His-tag-TTR-Lysozyme的构建：合成如SEQ ID NO:6所示的His-tag-TTR-Lysozyme序列，将其用Nde I和EcoR I酶与pET 21a(+)连接，经测序验证，构建成功。
(2)TTR-Lysozyme融合蛋白的表达和纯化：将步骤(1)构建的pET 21a(+)-His-tag-TTR-Lysozyme质粒转化入E.coli BL21(DE3)细胞，将所得重组E.coli BL21(DE3)在LB培养基中培养，制备种子液，以5％的接种量，接入5L TB培养基中，温度37℃，搅拌桨转速150rpm，培养OD 600至1.5-2.0；加入0.1-0.5mM IPTG进行诱导8-16h；利用高压均质破菌并将上清液通过镍柱亲和吸附制备得到TTR-Lysozyme融合蛋白。所得蛋白通过内毒素吸附柱(Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，通过0.22μm孔径滤膜去除残留菌体。对TTR-Lysozyme蛋白产量进行检测，结果如表3所示。
表3不同诱导条件下TTR-Lysozyme的表达
Figure PCTCN2020128588-appb-000009
Figure PCTCN2020128588-appb-000010
实施例4：转甲状腺素蛋白-鸡蛋清白蛋白的融合蛋白的制备
转甲状腺素蛋白-鸡蛋清白蛋白的融合蛋白(TTR-Ovalbumin)按照如下方法制备：
(1)重组质粒pET 21a(+)-His-tag-TTR-Ovalbumin的构建：合成如SEQ ID NO:7所示的His-tag-TTR-Ovalbumin序列，将其用Nde I和EcoR I酶与pET 21a(+)连接，经测序验证，构建成功。
(2)TTR-Ovalbumin融合蛋白的表达和纯化：将步骤(1)构建的pET 21a(+)-His-tag-TTR-Ovalbumin质粒转化至E.coli BL21(DE3)细胞，将所得重组E.coli BL21(DE3)在LB培养基中培养，制备种子液，以5％的接种量，接入5L TB培养基中，温度37℃，搅拌桨转速150rpm，培养OD 600至1.5-2.0；加入0.1-0.5mM IPTG进行诱导8-16h；利用高压均质破菌并将上清液通过镍柱亲和吸附制备得到TTR-Ovalbumin融合蛋白。所得蛋白通过内毒素吸附柱(Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，通过0.22μm孔径滤膜去除残留菌体。对TTR-Ovalbumin蛋白产量进行检测，结果如表4所示。
表4不同诱导条件下TTR-Ovalbumin的表达
Figure PCTCN2020128588-appb-000011
Figure PCTCN2020128588-appb-000012
图5中显示了E.coli BL21(DE3)表达转甲状腺素蛋白，融合蛋白纯化产物以及绿色荧光蛋白、鸡蛋清溶菌酶与鸡蛋清白蛋白标准品的电泳图谱。由图中可知，上述这些蛋白均正确表达。
实施例5：人源转甲状腺素蛋白的重组制备
(1)重组质粒pETx-rhaPBAD-ttr的构建：对pET-21a质粒(购于ATCC中国菌种保藏中心)进行改造重构(重构后所得质粒与原始pET-21a质粒在序列上的差别在约75％左右，具体序列如SEQ ID NO:8所示)，利用rhaPBAD启动子(鼠李唐诱导型)替代T7启动子，同时连接人源TTR优化核酸序列(如SEQ ID NO:2所示，TTR的氨基酸序列如SEQ ID NO:1所示)，所得质粒整体的核酸序列如SEQ ID NO:3所示。经测序验证(测序公司为南京金斯瑞生物科技有限公司)，构建成功。
(2)重组人源TTR的表达和纯化：将步骤(1)构建的pETx-rhaPBAD-ttr质粒转化入E.coli BL21(DE3)细胞，将所得重组E.coli BL21(DE3)在LB培养基中培养，制备种子液，再以5％的接种量，接入至5L TB培养基中，温度37℃，搅拌桨转速150rpm，培养至OD 600为1.5-2.0；加入0.4-2％(质量体积百分比)鼠李糖诱导16-20h(表5)。通过高压均质机将菌体破碎后，上清液通过镍(Ni +)柱层析制备得到人源TTR。使用内毒素吸附柱(Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，通过0.22μm孔径滤膜去除残留菌体。所得人源TTR蛋白产量如表5所示。在OD 600为1.8-2.0时，以1.6-2％鼠李糖诱导18-19h，可获得的蛋白产量≥50mg/g湿菌体。
表5人源TTR的重组表达
Figure PCTCN2020128588-appb-000013
Figure PCTCN2020128588-appb-000014
(3)重组大鼠源TTR和小鼠源TTR的表达和纯化同上所述，仅将人源TTR优化核酸序列替换成对应的鼠源TTR核酸序列，其余的步骤均相同。其中，大鼠源TTR的氨基酸序列如SEQ ID NO:9所示，小鼠源TTR的氨基酸序列如SEQ ID NO:10所示。
(4)人源成熟TTR丢失C末端-TNPKE后的蛋白质产物依据上述重组表达的步骤纯化得到，其产物命名为人源TTR-CL，氨基酸序列如SEQ ID NO:11所示。
(5)化学修饰人源TTR：设计并合成一段化学修饰基团，其是由马来酰亚胺、正十二烷以及5-氨基荧光素连接而成的疏水性修饰片段(Ex 490nm，Em 520nm)，然后将该化学修饰基团与上述重组表达及纯化的人源TTR中唯一的半胱氨酸(C)残基进行靶向的化学修饰。在4℃情况下，人源TTR与化学修饰基团以1：5的摩尔比例缓慢震荡反应(反应过程如图12所示)。反应终止后，通过超滤去除残留的化学修饰试剂并对TTR进行浓缩，样品通过荧光光谱仪检测，在490nm波长下激发，发射波长为520nm，说明成功对TTR的唯一半胱氨酸残基进行了靶向修饰，将其命名为人源TTR-Modified。
实施例6：人源TTR核酸序列优化前后的对比
将实施例5中改造重构后的pET-21a质粒上面的T7启动子利用rhaPBAD启动子(鼠李唐诱导型)进行替代，与核酸序列未优化的人源TTR连接，构建得到重组质粒pETx-rhaPBAD-ttr(未优化)，即所得重组质粒与实施例5中重组质粒pETx-rhaPBAD-ttr的区别仅在于TTR是否经过优化。再使用与实施例5第(2)部分相同的方法进行重组人源TTR的表达和纯化，所得结果如下表6所示。由表中可以看出，TTR蛋白均有表达，且在OD 600为1.6-2.0时，以1.2～2％鼠李糖诱导17～20h，可获得的蛋白产量≥17mg/g湿菌体，蛋白表达量较高。
表6人源TTR的重组表达(pETx-rhaPBAD-ttr(未优化))
Figure PCTCN2020128588-appb-000015
Figure PCTCN2020128588-appb-000016
第二部分：TTR本身及与蛋白融合后可以通过角膜屏障进入玻璃体腔及眼底
实施例7：人源TTR滴眼方式跨越角膜屏障进入玻璃体腔及眼底
(1)将实施例5所得的人源TTR配置为10μmol/L(含有生理盐水)，分别对C57BL/6小鼠(购于上海实验动物研究中心，8周龄)及SD大鼠(购于上海实验动物研究中心，8周龄)进行滴眼，滴入一滴(～30μl)，等待3h后处死，分别取下角膜、玻璃体与眼底样本(视网膜，脉络膜)，并分别提取蛋白。由于重组人源TTR带有His-tag标签，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过western-blot，测定C57BL/6小鼠及SD大鼠的角膜及玻璃体与眼底样本中，是否存在重组的人源TTR。结果显示，角膜组织提取蛋白中，信号不显著，而玻璃体与眼底样本中信号显著(图6)，说明通过滴眼方式，TTR可跨越角膜屏障进入玻璃体及眼底。
(2)将实施例5所得的人源TTR配置为5-30μmol/L(含有生理盐水，以及0-6％低分子量透明质酸)，分别对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼，经过3-72h后处死，取玻璃体与眼底样本提取蛋白，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过ELISA，测定C57BL/6小鼠及SD大鼠的玻璃体与眼底样本中，人源TTR的含量。结果显示，滴眼液中TTR含量为10-15μmol/L时，滴眼3h后玻璃体与眼底样本中人源TTR含量达到最高峰；TTR浓度达到15μmol/L后，再进一步增加TTR的浓度对增加玻璃体与眼底样本中人源TTR浓度的作用不大；滴眼液中，透明质酸含量为2％时，滴眼3h后玻璃体与眼底样本中人源TTR含量达到最高峰；滴眼后，人源TTR在C57BL/6小鼠及SD大鼠玻璃体与眼底样本内的含量半衰期接近60h，说明可以在玻璃体与眼底样本内有效存在60h，有足够的治疗浓度和治疗时间(表7-1)。
表7-1人源TTR跨越C57BL/6小鼠及SD大鼠角膜屏障进入玻璃体腔及眼底
Figure PCTCN2020128588-appb-000017
Figure PCTCN2020128588-appb-000018
Figure PCTCN2020128588-appb-000019
(3)将实施例5所得的人源TTR以及人源TTR-Modified配置为10μmol/L(含有生理盐水，以及2％低分子量透明质酸)，分别对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼，经过3-72h后处死，取玻璃体与眼底样本(视网膜，脉络膜)提取蛋白，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过ELISA，测定C57BL/6小鼠及SD大鼠的玻璃体与眼底样本中，人源TTR的含量。结果显示，人源TTR-Modified经过长疏水片段修饰后，进入玻璃体及眼底的效率显著高于未修饰的人源TTR(表7-2)。
表7-2人源TTR及人源TTR-Modified进入玻璃体腔及眼底效率比对
Figure PCTCN2020128588-appb-000020
Figure PCTCN2020128588-appb-000021
实施例8：TTR滴眼方式跨越角膜屏障进入玻璃体腔
将实施例1制备的去除内毒素及细菌后的纯化的TTR蛋白(浓度为4μmol/L)，通过滴眼的方式处理健康SD大鼠(rattus norregicus)(6周龄)及新西兰大耳兔(Oryctolagus cuniculus)(2月龄，～2.5kg)，左眼为滴加蛋白样本眼，右眼滴加生理盐水作为空白对照。每日滴加次数为1-3次，每次滴加量为0.4-0.8nmol。两周后摘取眼球并分离获得玻璃体样本，通过western-blot法初步验证(图7A)TTR能够从眼表进入玻璃体腔，以Anti-His tag抗体为一抗的western-blot结果显示SD大鼠左眼玻璃体内外源TTR的信号强度为右眼的28.7倍，新西兰大耳兔左眼玻璃体内外源TTR的信号强度为右眼的35.6倍；利用ELISA法，以Anti-His tag抗体为一抗测定玻璃体样本内TTR的含量(表8，9)。结果显示，每日以0.6nmol含量滴加2次后，SD大鼠及新西兰大耳兔玻璃体内检测到较高值的外源TTR含量。
此外，图3中显示了人/大鼠/小鼠/家兔源转甲状腺素蛋白经同源性比对，序列相似性达到近95％，可见图7A中的本体TTR阳性信号是眼内的同源蛋白信号。
表8不同处理方式下TTR到达SD大鼠眼内的效果
Figure PCTCN2020128588-appb-000022
Figure PCTCN2020128588-appb-000023
表9不同处理方式下TTR到达新西兰大耳兔眼内的效果
Figure PCTCN2020128588-appb-000024
实施例9：TTR-GFP融合蛋白滴眼方式跨越角膜屏障进入玻璃体腔
将实施例2制备的去除内毒素及细菌后的纯化的TTR-GFP蛋白(浓度为4μmol/L)，通过滴眼的方式处理健康SD大鼠(rattus norregicus)(6周龄)及新西兰大耳兔(Oryctolagus cuniculus)(2月龄，～2.5kg)，左眼为滴加蛋白样本眼，右眼滴加生理盐水作为空白对照。每日滴加次数为1-3次，每次滴加量为0.4-0.8nmol。两周后摘取眼球并分离获得玻璃体样本，通过western-blot法初步验证(图7B)TTR-GFP能够从眼表进入玻璃体腔，以Anti-GFP抗体为一抗的western-blot结果显示SD大鼠左眼玻璃体内外源GFP的信号强度为右眼的62.3倍，新西兰大耳兔左眼玻璃体内外源GFP的信号强度为右眼的47.6倍；以Anti-His tag抗体为一抗的western-blot结果显示SD大鼠左眼玻璃体内外源TTR的信号强度较强而右眼无信号，新西兰大耳兔左眼玻璃体内外源TTR的信号强度为右眼的45.4倍。利用ELISA法以Anti-His tag抗体为一抗测定玻璃体样本内TTR-GFP的含量(表10，11)。结果显示，每日以0.6nmol含量滴加2次后，SD大鼠及新西兰大耳兔玻璃体内检测到较高值的外源TTR-GFP含量。
表10不同处理方式下TTR-GFP到达SD大鼠眼内的效果
Figure PCTCN2020128588-appb-000025
Figure PCTCN2020128588-appb-000026
表11不同处理方式下TTR-GFP到达新西兰大耳兔眼内的效果
Figure PCTCN2020128588-appb-000027
实施例10：TTR-Lysozyme融合蛋白滴眼方式跨越角膜屏障进入玻璃体腔
将实施例3制备的去除内毒素及细菌后的纯化的TTR-Lysozyme蛋白(浓度为4μmol/L)，通过滴眼的方式处理健康SD大鼠(rattus norregicus)(6周龄)及新西兰大耳兔(Oryctolagus cuniculus)(2月龄，～2.5kg)，左眼为滴加蛋白样本眼，右眼滴加生理盐水作为空白对照。每日滴加次数为1-3次，每次滴加量为0.4-0.8nmol。两周后摘取眼球并分离获得玻璃体样本，通过western-blot法初步验证(图7C)TTR-Lysozyme能够从 眼表进入玻璃体腔，以Anti-Lysozyme抗体为一抗的western-blot结果显示SD大鼠左眼玻璃体内外源Lysozyme的信号强度为右眼的34.6倍，新西兰大耳兔左眼玻璃体内外源Lysozyme的信号强度较强而右眼无信号；以Anti-His tag抗体为一抗的western-blot结果显示SD大鼠左眼玻璃体内外源TTR的信号强度为右眼的30.2倍，新西兰大耳兔左眼玻璃体内外源TTR的信号强度为右眼的46.3倍。利用ELISA法以Anti-His tag抗体为一抗测定玻璃体样本内TTR-Lysozyme的含量(表12，13)。结果显示，每日以0.6nmol含量滴加2次后，SD大鼠及新西兰大耳兔玻璃体内检测到较高值的外源TTR-Lysozyme含量。
表12不同处理方式下TTR-Lysozyme到达SD大鼠眼内的效果
Figure PCTCN2020128588-appb-000028
表13不同处理方式下TTR-Lysozyme到达新西兰大耳兔眼内的效果
Figure PCTCN2020128588-appb-000029
Figure PCTCN2020128588-appb-000030
实施例11：TTR-Ovalbumin融合蛋白滴眼方式跨越角膜屏障进入玻璃体腔
将实施例4制备的去除内毒素及细菌后的纯化的TTR-Ovalbumin蛋白(浓度为4μmol/L)，通过滴眼的方式处理健康SD大鼠(rattus norregicus)(6周龄)及新西兰大耳兔(Oryctolagus cuniculus)(2月龄，～2.5kg)，左眼为滴加蛋白样本眼，右眼滴加生理盐水作为空白对照。每日滴加次数为1-3次，每次滴加量为0.4-0.8nmol。两周后摘取眼球并分离获得玻璃体样本，通过western-blot法初步验证(图7D)TTR-Ovalbumin能够从眼表进入玻璃体腔，以Anti-Ovalbumin抗体为一抗的western-blot结果显示SD大鼠左眼玻璃体内外源Ovalbumin的信号强度为右眼的25.3倍，新西兰大耳兔左眼玻璃体内外源Ovalbumin的信号较强而右眼无信号；以Anti-His tag抗体为一抗的western-blot结果显示SD大鼠左眼玻璃体内外源TTR的信号信号强度为右眼的37.8倍，新西兰大耳兔左眼玻璃体内外源TTR的信号强度较强而右眼无信号。利用ELISA法以Anti-His tag抗体为一抗测定玻璃体样本内TTR-Ovalbumin的含量(表14，15)。结果显示，每日以0.6nmol含量滴加2次后，SD大鼠及新西兰大耳兔玻璃体内检测到较高值的外源TTR-Ovalbumin含量。
表14不同处理方式下TTR-Ovalbumin到达SD大鼠眼内的效果
Figure PCTCN2020128588-appb-000031
Figure PCTCN2020128588-appb-000032
表15不同处理方式下TTR-Ovalbumin到达新西兰大耳兔眼内的效果
Figure PCTCN2020128588-appb-000033
第三部分：TTR治疗糖网等眼部疾病
实施例12：人源TTR滴眼方式治疗DR(糖尿病视网膜病变)SD大鼠
8周龄SD大鼠，体重200-250g，禁食12-18h，腹腔注射2％STZ(60mg/kg)，48h及72h后剪尾采血，血糖试纸检测均高于16.7mM，造模成功，获得DR SD大鼠。DR SD大鼠分为2大组，1组为DR SD大鼠不加任何处理(5只)，另外1组为实施例5所制得的人源TTR滴眼组，25只，左眼每日滴加2次人源TTR(5-20μmol/L)(生理盐水+2％透明质酸)，右眼滴加生理盐水+2％透明质酸；此外，另有1组正常SD大鼠(未滴眼)作为正常对照组(5只)。所有SD大鼠继续饲养3个月后，分别剥取视网膜进行Evans Blue(EB)染色观察视网膜血管渗漏情况，Trypsin酶解观察新生血管密度；结果显示，STZ诱导SD大鼠饲养3个月后，与正常对照相比，视网膜血管渗漏，新生血管数量均显著提升，而滴加人源TTR的眼球视网膜渗漏现象显著抑制，视网膜新生血管数显著下降(图8)，说明了DR的临床病理现象得到了缓解。其中，每日滴加10μmol/L人源TTR(生理盐水+2％透明质酸)的效果最佳(表16)。
表16人源TTR治疗STZ诱导SD大鼠DR病理状况
Figure PCTCN2020128588-appb-000034
实施例13：人源TTR滴眼方式治疗ROP(早产儿视网膜病变)SD大鼠
出生一周乳鼠(SD大鼠)，放入高压氧舱饲养，正常对照组放入正常环境饲养(正常对照组，6只)。高压氧舱中，一组乳鼠使用5-20μmol/L实施例5所制得的TTR(生理盐水+2％透明质酸)滴眼，每天滴1次，每次30μl(ROP/TTR(造模)组，6只)；高压氧舱中，另一组乳鼠不处理(ROP组，24只)；高压氧舱饲养5天后，将所有乳鼠从高压氧舱转移至正常环境饲养，并在ROP组中分出一组每日使用5-20μmol/L实施例5所制得的TTR(生理盐水+2％透明质酸)滴眼，每天滴1次，每次30μl(ROP/TTR(成模)组)；而ROP组其余乳鼠则不加任何处理作为对照；正常环境饲养5天后处死乳鼠并剥取视网膜进行EB染色。
结果如图9所示，图中显示，造模过程中，正常对照组视网膜EB染色形态正常；ROP组存在显著无灌注区及新生血管；而造模同时TTR滴眼的ROP/TTR(造模)组则均没有形成显著的无灌注区及畸形新生血管，这说明在缺氧状态下TTR对正常血管有保护作用，对新生血管具有抑制作用(图9)。
对已成模的ROP乳鼠进行TTR(10μmol/L)滴眼，持续5天。比较ROP及ROP/TTR(成模)组，在实验后期(5天)，ROP组内畸形新生血管布满视网膜后，出现大量的渗漏区域，而TTR滴眼能够逆转这种趋势(图10)。
不同浓度TTR(5-20μmol/L)对已成模的ROP乳鼠进行滴眼，持续5天。在实验后期(5天)，ROP对照组内畸形新生血管布满视网膜，出现大量的渗漏区域，而TTR滴眼能够逆转这种趋势，其中10μmol/L TTR滴加量效果最佳(表17)。
表17人源TTR治疗高压氧舱诱导SD大鼠乳鼠ROP病理状况
Figure PCTCN2020128588-appb-000035
实施例14：人源TTR滴眼方式治疗AMD(老年性黄斑变性)C57BL/6小鼠
9周龄C57/BL6小鼠，用氪激光(647nm)对视网膜进行光凝，功率360mW，直径50μm，时间0.05s，每眼8个光凝点，诱导脉络膜新生血管生成，并逐步向视网膜增生移行，获得AMD C57BL/6小鼠。AMD C57/BL6小鼠分为滴眼组及未滴眼组，滴眼组14只，进行TTR滴眼，每日右眼滴加5-20μmol/L实施例5所制得的TTR(生理盐水+2％透明质酸)两次，每次30μL，左眼滴加生理盐水+2％透明质酸作为对照；未滴眼组6只，不做任何处理；另有未打激光组2只(未滴眼)作为正常对照组；滴眼2周后，处死动物，剥离视网膜并进行EB染色观察视网膜血管渗漏情况，以及Trypsin酶解观察新生血管密度。
结果如图11所示，图中显示，正常对照组视网膜EB染色形态正常；AMD组存在显著视网膜渗漏及新生血管；而10μmol/L TTR滴眼的AMD/TTR组视网膜渗漏情况及新生血管情况得到显著的缓解(图11)。
不同浓度TTR(5-20μmol/L)对已成模的AMD小鼠进行滴眼，持续两周。AMD对照组内出现较多的渗漏区域，新生血管数量显著增多，而TTR滴眼能够逆转这种趋势，其中10μmol/L TTR滴加量效果最佳(表18)。
表18人源TTR治疗激光视网膜光凝诱导C57BL/6小鼠AMD病理状况
Figure PCTCN2020128588-appb-000036
由以上实施例可知，通过TTR滴眼的方式，在DR、AMD以及ROP的动物模型中，能够有效治疗DR、AMD以及ROP的病理状态。
实施例15：人源TTR/大鼠源TTR滴眼方式治疗DR(糖尿病视网膜病变)SD大鼠8周龄SD大鼠，体重200-250g，禁食12-18h，腹腔注射2％STZ(60mg/kg)，48h及72h后剪尾采血，血糖试纸检测均高于16.7mM，造模成功，获得DR SD大鼠。DR SD大鼠分为5大组，1组为DR SD大鼠不加任何处理(5只)；1组为实施例5所制得的人源TTR滴眼组，5只，左眼每日滴加2次人源TTR(10μmol/L)(生理盐水+2％透明质酸)，右眼滴加生理盐水+2％透明质酸；1组为实施例5所制得的大鼠源TTR滴眼组，5只，左眼每日滴加2次实施例5所制得的大鼠源TTR(10μmol/L)(生理盐水+2％ 透明质酸)，右眼滴加生理盐水+2％透明质酸；1组为实施例5所制得的人源TTR-CL滴眼组，5只，左眼每日滴加2次实施例5所制得的人源TTR-CL(10μmol/L)(生理盐水+2％透明质酸)，右眼滴加生理盐水+2％透明质酸；最后1组为实施例5所制得的人源TTR-Modified滴眼组，5只，左眼每日滴加2次实施例5所制得的人源TTR-Modified(10μmol/L)(生理盐水+2％透明质酸)，右眼滴加生理盐水+2％透明质酸；另有1组正常SD大鼠(未滴眼)作为正常对照组(5只)。所有SD大鼠继续饲养3个月后，分别剥取视网膜进行EB染色观察视网膜血管渗漏情况，Trypsin酶解观察新生血管密度；结果显示，STZ诱导SD大鼠饲养3个月后，与正常对照相比，视网膜血管渗漏，新生血管数量均显著提升，而滴加人源TTR、人源TTR-CL、人源TTR-Modified以及大鼠源TTR的眼球视网膜渗漏现象显著抑制，视网膜新生血管数显著下降，说明了DR的临床病理现象得到了缓解。
表19人源TTR/大鼠源TTR治疗STZ诱导SD大鼠DR病理状况
Figure PCTCN2020128588-appb-000037
实施例16：人源TTR/大鼠源TTR滴眼方式治疗ROP(早产儿视网膜病变)SD大鼠
出生一周乳鼠(SD大鼠)，放入高压氧舱饲养；高压氧舱饲养5天后，造模成功，将所有乳鼠从高压氧舱转移至正常环境饲养，对部分已成模的ROP乳鼠进行TTR(10μmol/L)滴眼，每日一次，持续5天。比较ROP未滴眼组(5只)，ROP+人源TTR滴眼组(5只)，ROP+人源TTR-CL滴眼组(5只)，ROP+人源TTR-Modified滴眼组(5只)，ROP+大鼠源TTR滴眼组(5只)，另有非ROP模型大鼠(未滴眼)作为正常对照组(5只)。ROP组内畸形新生血管布满视网膜后，出现大量的渗漏区域，而TTR滴眼能够逆转这种趋势。
表20人源TTR/大鼠源TTR治疗高压氧舱诱导SD大鼠乳鼠ROP病理状况
Figure PCTCN2020128588-appb-000038
实施例17：人源TTR/小鼠源TTR滴眼方式治疗AMD(老年性黄斑变性)C57BL/6小鼠
9周龄C57/BL6小鼠，用氪激光(647nm)对视网膜进行光凝，功率360mW，直径50μm，时间0.05s，每眼8个光凝点，诱导脉络膜新生血管生成，并逐步向视网膜增生移行，获得AMD C57BL/6小鼠。AMD C57/BL6小鼠分为滴眼组及未滴眼组，滴眼组进行TTR滴眼，每日右眼滴加10μmol/L人源TTR或小鼠源TTR(生理盐水+2％透明质酸)两次，每次30μL，左眼滴加生理盐水+2％透明质酸作为对照，人源TTR滴眼组5只，人源TTR-CL滴眼组5只，人源TTR-Modified滴眼组5只，小鼠源TTR滴眼组5只；未滴眼组5只，不做任何处理；另有非激光光凝小鼠(未滴眼)作为正常对照组(5只)。滴眼2周后，处死动物，剥离视网膜并进行EB染色观察视网膜血管渗漏情况，以及Trypsin酶解观察新生血管密度。由表21中可知，滴加人源TTR、人源TTR-CL、人源TTR-Modified以及小鼠源TTR的眼球视网膜渗漏现象显著抑制，视网膜新生血管数显著下降，说明了AMD的临床病理现象得到了缓解。
表21人源TTR/小鼠源TTR治疗激光视网膜光凝诱导C57BL/6小鼠AMD病理状况
Figure PCTCN2020128588-appb-000039
Figure PCTCN2020128588-appb-000040
第四部分：辅料的改进
实施例18：使用羧甲基纤维素钠作为辅料可促进TTR通过角膜屏障
(1)将实施例5制得的人源TTR配置为5-30μmol/L(含有生理盐水)，分别对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼，经过3h后处死，取玻璃体与眼底样本提取蛋白，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过ELISA，测定C57BL/6小鼠及SD大鼠的玻璃体与眼底样本中人源TTR的含量。
(2)将实施例5制得的人源TTR配置为10μmol/L(含有生理盐水及0-8mg/mL羧甲基纤维素钠(购于国药，粘度为800-1200CP))，分别对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼，经过3-72h后处死，取玻璃体与眼底样本提取蛋白，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过ELISA，测定C57BL/6小鼠及SD大鼠的玻璃体与眼底样本中人源TTR的含量。
结果显示，滴眼3h后玻璃体与眼底样本中人源TTR含量达到最高峰；滴眼液中，添加了羧甲基纤维素钠之后，玻璃体与眼底样本中人源TTR的含量均显著提高(提高10％以上)，且当羧甲基纤维素钠含量为6mg/mL时，滴眼3h后玻璃体与眼底样本中人源TTR含量达到最高峰；滴眼后，人源TTR在C57BL/6小鼠及SD大鼠玻璃体与眼底样本内的含量半衰期接近72h，说明可以在玻璃体与眼底样本内有效存在72h，有足够的治疗浓度和治疗时间(表22)。
表22人源TTR跨越C57BL/6小鼠及SD大鼠角膜屏障进入玻璃体及眼底
Figure PCTCN2020128588-appb-000041
Figure PCTCN2020128588-appb-000042
Figure PCTCN2020128588-appb-000043
实施例19：使用右旋糖酐70作为辅料可促进TTR通过角膜屏障
(1)将实施例5制得的人源TTR配置为5-30μmol/L(含有生理盐水)，分别对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼，经过3h后处死，取玻璃体与眼底样本提取蛋白，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过ELISA，测定C57BL/6小鼠及SD大鼠的玻璃体与眼底样本中人源TTR的含量。
(2)将实施例5制得的人源TTR配置为10μmol/L(含有生理盐水及0-0.8mg/mL右旋糖酐70(购于国药，分子量为64000-76000))，分别对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼，经过3-72h后处死，取玻璃体与眼底样本提取蛋白，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过ELISA，测定C57BL/6小鼠及SD大鼠的玻璃体与眼底样本中人源TTR的含量。
结果显示，滴眼3h后玻璃体与眼底样本中人源TTR含量达到最高峰；滴眼液中，添加了右旋糖酐70之后，玻璃体与眼底样本中人源TTR的含量均显著提高(提高15％以上)，且当右旋糖酐70含量为0.4mg/mL时，滴眼3h后玻璃体与眼底样本中人源TTR含量达到最高峰；滴眼后，人源TTR在C57BL/6小鼠及SD大鼠玻璃体与眼底样本内的含量半衰期接近60h，说明可以在玻璃体与眼底样本内有效存在60h，有足够的治疗浓度和治疗时间(表23)。
表23人源TTR跨越C57BL/6小鼠及SD大鼠角膜屏障进入玻璃体及眼底
Figure PCTCN2020128588-appb-000044
Figure PCTCN2020128588-appb-000045
实施例20：使用硫酸软骨素A钠盐作为辅料可促进TTR通过角膜屏障
(1)将实施例5制得的人源TTR配置为5-30μmol/L(含有生理盐水)，分别对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼，经过3h后处死，取玻璃体与眼底样本提取蛋白，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过ELISA，测 定C57BL/6小鼠及SD大鼠的玻璃体与眼底样本中人源TTR的含量。
(2)将实施例5制得的人源TTR配置为10μmol/L(含有生理盐水及0-40mg/mL硫酸软骨素A钠盐(购于国药))，分别对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼，经过3-72h后处死，取玻璃体与眼底样本提取蛋白，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过ELISA，测定C57BL/6小鼠及SD大鼠的玻璃体与眼底样本中人源TTR的含量。
结果显示，滴眼3h后玻璃体与眼底样本中人源TTR含量达到最高峰；滴眼液中，添加了硫酸软骨素A钠盐之后，玻璃体与眼底样本中人源TTR的含量均显著提高(提高17％以上)，且当硫酸软骨素A钠盐含量为20mg/mL时，滴眼3h后玻璃体与眼底样本中人源TTR含量达到最高峰；滴眼后，人源TTR在C57BL/6小鼠及SD大鼠玻璃体与眼底样本内的含量半衰期接近72h，说明可以在玻璃体与眼底样本内有效存在72h，有足够的治疗浓度和治疗时间(表24)。
表24人源TTR跨越C57BL/6小鼠及SD大鼠角膜屏障进入玻璃体及眼底
Figure PCTCN2020128588-appb-000046
Figure PCTCN2020128588-appb-000047
实施例21：人源TTR-辅料滴眼方式治疗DR(糖尿病视网膜病变)SD大鼠
8周龄SD大鼠，体重200-250g，禁食12-18h，腹腔注射2％STZ(60mg/kg)，48h及72h后剪尾采血，血糖试纸检测均高于16.7mM，造模成功，获得DR SD大鼠。DR SD大鼠分为6组，1组为DR SD大鼠不加任何处理(5只)，另外5组均为5只/组，左眼、右眼各每日滴加2次，每次30μL，其中，左眼为采用实施例5所制得的人源TTR滴眼，或者采用实施例5所制得的人源TTR加辅料滴眼，具体为分别采用10μmol/L人源TTR(生理盐水溶液)、10μmol/L人源TTR(生理盐水溶液+6mg/mL羧甲基纤维素钠)、10μmol/L人源TTR(生理盐水溶液+6mg/mL PEG400)、10μmol/L人源TTR(生 理盐水溶液+0.4mg/mL右旋糖酐70)、10μmol/L人源TTR(生理盐水溶液+20mg/mL硫酸软骨素A钠盐)滴眼，右眼为采用生理盐水溶液，或者生理盐水溶液加辅料滴眼，以作为对照，具体为分别采用生理盐水溶液、生理盐水溶液+6mg/mL羧甲基纤维素钠、生理盐水溶液+6mg/mL PEG400、生理盐水溶液+0.4mg/mL右旋糖酐70、生理盐水溶液+20mg/mL硫酸软骨素A钠盐滴眼。此外，另有1组正常SD大鼠作为对照(5只)。所有SD大鼠继续饲养3个月后，分别剥取视网膜进行EB染色观察视网膜血管渗漏情况，Trypsin酶解观察新生血管密度。结果显示，STZ诱导SD大鼠饲养3个月后，与正常对照相比，视网膜血管渗漏、新生血管数量均显著提升，而滴加人源TTR的眼球视网膜渗漏现象显著抑制，视网膜新生血管数显著下降，说明了DR的临床病理现象得到了缓解。其中，每日滴加10μmol/L人源TTR(生理盐水溶液+20mg/mL硫酸软骨素A钠盐)的效果最佳(表25)。而滴加10μmol/L人源TTR(生理盐水溶液+6mg/mL PEG400)的治疗效果相对较差。
表25人源TTR/人源TTR-辅料治疗STZ诱导SD大鼠DR病理状况
Figure PCTCN2020128588-appb-000048
Figure PCTCN2020128588-appb-000049
实施例22：人源TTR-辅料滴眼方式治疗AMD(老年性黄斑变性)C57BL/6小鼠
9周龄C57/BL6小鼠，用氪激光(647nm)对视网膜进行光凝，功率360mW，直径50μm，时间0.05s，每眼8个光凝点，诱导脉络膜新生血管生成，并逐步向视网膜增生移行，获得AMD C57BL/6小鼠。AMD C57BL/6小鼠分为6组，1组为AMD C57BL/6小鼠不加任何处理(5只)，另外5组均为5只/组，左眼、右眼各每日滴加2次，每次30μL，其中，左眼为采用实施例5所制得的人源TTR滴眼，或者采用实施例5所制得 的人源TTR加辅料滴眼，具体为分别采用10μmol/L人源TTR(生理盐水溶液)、10μmol/L人源TTR(生理盐水溶液+6mg/mL羧甲基纤维素钠)、10μmol/L人源TTR(生理盐水溶液+6mg/mL PEG400)、10μmol/L人源TTR(生理盐水溶液+0.4mg/mL右旋糖酐70)、10μmol/L人源TTR(生理盐水溶液+20mg/mL硫酸软骨素A钠盐)滴眼，右眼为采用生理盐水溶液，或者生理盐水溶液加辅料滴眼，以作为对照，具体为分别采用生理盐水溶液、生理盐水溶液+6mg/mL羧甲基纤维素钠、生理盐水溶液+6mg/mL PEG400、生理盐水溶液+0.4mg/mL右旋糖酐70、生理盐水溶液+20mg/mL硫酸软骨素A钠盐滴眼。此外，另有1组正常C57BL/6小鼠作为对照(5只)。滴眼2周后，处死动物，剥离视网膜并进行EB染色观察视网膜血管渗漏情况，以及Trypsin酶解观察新生血管密度。
结果显示，AMD C57BL/6小鼠对照组内出现较多的渗漏区域，新生血管数量显著增多，而TTR滴眼能够逆转这种趋势(表26)，且添加了羧甲基纤维素钠、右旋糖酐70和硫酸软骨素A钠盐的组中，TTR的治疗效果更佳。
表26人源TTR/人源TTR-辅料治疗激光视网膜光凝诱导C57BL/6小鼠AMD病理状况
Figure PCTCN2020128588-appb-000050
Figure PCTCN2020128588-appb-000051
实施例23：人源TTR-辅料滴眼方式治疗ROP(早产儿视网膜病变)SD大鼠
出生一周乳鼠(SD大鼠)，放入高压氧舱饲养，正常对照组放入正常环境饲养(正常对照组，5只)。高压氧舱饲养5天后取出，获得ROP SD大鼠。将所有ROP SD大鼠连同正常对照组一起在正常环境中继续饲养5天。在该5天中，ROP SD大鼠分为6组，1组为ROP SD小鼠不加任何处理(5只)，另外5组均为5只/组，左眼、右眼各每日滴加1次，每次30μL，其中，左眼为采用实施例5所制得的人源TTR滴眼，或者采用实 施例5所制得的人源TTR加辅料滴眼，具体为分别采用10μmol/L人源TTR(生理盐水溶液)、10μmol/L人源TTR(生理盐水溶液+6mg/mL羧甲基纤维素钠)、10μmol/L人源TTR(生理盐水溶液+6mg/mL PEG400)、10μmol/L人源TTR(生理盐水溶液+0.4mg/mL右旋糖酐70)、10μmol/L人源TTR(生理盐水溶液+20mg/mL硫酸软骨素A钠盐)滴眼，右眼为采用生理盐水溶液，或者生理盐水溶液加辅料滴眼，以作为对照，具体为分别采用生理盐水溶液、生理盐水溶液+6mg/mL羧甲基纤维素钠、生理盐水溶液+6mg/mL PEG400、生理盐水溶液+0.4mg/mL右旋糖酐70、生理盐水溶液+20mg/mL硫酸软骨素A钠盐滴眼。滴眼5天后，处死动物，剥离视网膜并进行EB染色观察视网膜血管渗漏情况。ROP SD大鼠对照组内畸形新生血管布满视网膜后，出现大量的渗漏区域，而TTR滴眼能够逆转这种趋势(表27)，且添加了羧甲基纤维素钠、右旋糖酐70和硫酸软骨素A钠盐的组中，TTR的治疗效果更佳。
表27人源TTR/人源TTR-辅料治疗高压氧舱诱导SD大鼠乳鼠ROP病理状况
Figure PCTCN2020128588-appb-000052
Figure PCTCN2020128588-appb-000053
第五部分：配体的改进
实施例24：计算机模拟TTR与各个配体分子的结合形态
参照PDB数据库中，TTR二聚体与双氯芬酸的静态共结晶模型(PDF：3CFQ)，如图13A所示。图13A中，蛋白结构为TTR二聚体，双氯芬酸配体分子使用箭头指示，一分子TTR二聚体可以结合两分子双氯芬酸。图13B中显示了双氯芬酸与TTR氨基酸残基的相互作用。
通过Discovery studio软件进行分子模拟发现维生素A1(视黄醇)能够与TTR多聚体的疏水通道稳定结合。图14A中显示的蛋白结构为TTR二聚体，维生素A1配体分子使用箭头指示，一分子TTR二聚体可以结合一分子维生素A1。图14B中显示了维生素A1与TTR氨基酸残基的相互作用。
通过Discovery studio软件进行分子模拟发现维生素A2(3-脱氢视黄醇)能够与TTR多聚体的疏水通道稳定结合。图15A中显示的蛋白结构为TTR二聚体，维生素A2配体分子使用箭头指示，一分子TTR二聚体可以结合一分子维生素A2。图15B中显示了维 生素A2与TTR氨基酸残基的相互作用。
通过Discovery studio软件进行分子模拟发现木犀草苷能够与TTR多聚体稳定结合。图16A中显示的蛋白结构为TTR二聚体，木犀草苷配体分子使用箭头指示，一分子TTR二聚体可以结合一分子木犀草苷。图16B中显示了木犀草苷与TTR氨基酸残基的相互作用。
从以上结果可以看出，TTR与这些配体分子之间的作用力主要为范德华力、氢键作用、疏水作用等非共价作用力，从本质上说不会改变TTR和配体之间的化学结构。
实施例25：TTR与各个潜在配体分子动态特异性结合参数的测定
使用nano ITC(TA)测定TTR与实施例24中所提及的各个配体分子或其盐的亲和结合平衡解离常数K d，在10μmol/L的TTR溶液(1000μL)中，以1μL/min的速度滴加100μmol/L的上述各种配体溶液，使用内置软件计算亲和结合平衡解离常数K d(表28)。
表28各种配体分子与TTR的亲和结合平衡解离常数
Figure PCTCN2020128588-appb-000054
如表28所示，各种配体分子与TTR的亲和结合平衡解离常数均接近10 -8mol/L，与单克隆抗体识别单一抗原表位的能力接近，说明上述配体分子能与TTR特异性识别与结合。
实施例26：人源TTR-配体分子复合物跨越角膜屏障进入玻璃体及眼底
将实施例5中所制得的人源TTR配置为10μmol/L(含有生理盐水，2％低分子量透明质酸以及10μmol/L双氯芬酸钠/5μmol/L维生素A+5％(v/v)吐温80/5μmol/L木犀草苷(添加比例参照实施例24中计算机模拟的结果)，分别对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼，经过3-72h后处死，取玻璃体与眼底样本提取蛋白，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过ELISA，测定C57BL/6小鼠及SD大 鼠的玻璃体与眼底样本中人源TTR的含量。参照中国药典2020年版第二部第115页，双氯芬酸钠滴眼液条目，测定样品中双氯芬酸钠含量；参照中国药典2020版第二部第1472页，维生素A条目，测定样品中维生素A含量；参照英国药典BP2017，Luteolin-7-glucoside条目，测定样品中木犀草苷含量。
滴眼后，在C57BL/6小鼠及SD大鼠玻璃体与眼底样本内的人源TTR以及各配体分子的含量半衰期接近60h，说明可以在玻璃体与眼底样本内有效存在60h，有足够的治疗浓度和治疗时间(表29)。
表29人源TTR及配体分子跨越C57BL/6小鼠及SD大鼠角膜屏障进入玻璃体及眼底
Figure PCTCN2020128588-appb-000055
Figure PCTCN2020128588-appb-000056
Figure PCTCN2020128588-appb-000057
实施例27：人源TTR-配体分子复合物滴眼方式治疗DR(糖尿病视网膜病变)SD大鼠
8周龄SD大鼠，体重200-250g，禁食12-18h，腹腔注射2％STZ(60mg/kg)，48h及72h后剪尾采血，血糖试纸检测均高于16.7mM，造模成功，获得DR SD大鼠。DR SD大鼠分为5组，1组为DR SD大鼠不加任何处理(5只)，另外4组均为5只/组，左眼、右眼各每日滴加2次，每次30μL，其中，左眼为采用实施例5所制得的人源TTR 滴眼，或者采用实施例5所制得的人源TTR+配体滴眼，具体为分别采用10μmol/L人源TTR(生理盐水溶液+2％透明质酸)，10μmol/L人源TTR(生理盐水溶液+2％透明质酸+10μmol/L双氯芬酸钠)、10μmol/L人源TTR(生理盐水溶液+2％透明质酸+5μmol/L维生素A+5％吐温80)、10μmol/L人源TTR(生理盐水溶液+2％透明质酸+5μmol/L木犀草苷)滴眼，右眼为采用生理盐水溶液+2％透明质酸，或者生理盐水溶液+2％透明质酸+配体滴眼，以作为对照，具体为分别采用生理盐水溶液+2％透明质酸，生理盐水溶液+2％透明质酸+10μmol/L双氯芬酸钠，生理盐水溶液+2％透明质酸+5μmol/L维生素A+5％吐温80，生理盐水溶液+2％透明质酸+5μmol/L木犀草苷滴眼。此外，另有1组正常SD大鼠作为对照(5只)。所有SD大鼠继续饲养3个月后，分别剥取视网膜进行EB染色观察视网膜血管渗漏情况，Trypsin酶解观察新生血管密度。
结果显示，STZ诱导SD大鼠饲养3个月后，与正常对照相比，DR SD大鼠不加任何处理的对照组的视网膜血管渗漏、新生血管数量均显著提升，而滴加人源TTR或者人源TTR/双氯芬酸钠、人源TTR/维生素A、人源TTR/木犀草苷组的眼球视网膜渗漏现象显著抑制，视网膜新生血管数显著下降，说明了DR的临床病理现象得到了缓解(表30)。
表30人源TTR/人源TTR-配体分子复合物治疗STZ诱导SD大鼠DR病理状况
Figure PCTCN2020128588-appb-000058
实施例28：人源TTR-配体分子复合物滴眼方式治疗AMD(老年性黄斑变性) C57BL/6小鼠
9周龄C57/BL6小鼠，用氪激光(647nm)对视网膜进行光凝，功率360mW，直径50μm，时间0.05s，每眼8个光凝点，诱导脉络膜新生血管生成，并逐步向视网膜增生移行，获得AMD C57BL/6小鼠。AMD C57BL/6小鼠分为5组，1组为AMD C57BL/6小鼠不加任何处理(5只)，另外4组均为5只/组，左眼、右眼各每日滴加2次，每次30μL，其中，左眼为采用实施例5所制得的人源TTR滴眼，或者采用实施例5所制得的人源TTR+配体滴眼，具体为分别采用10μmol/L人源TTR(生理盐水溶液+2％透明质酸)、10μmol/L人源TTR(生理盐水溶液+2％透明质酸+10μmol/L双氯芬酸钠)、10μmol/L人源TTR(生理盐水溶液+2％透明质酸+5μmol/L维生素A+5％吐温80)、10μmol/L人源TTR(生理盐水溶液+2％透明质酸+5μmol/L木犀草苷)滴眼，右眼为采用生理盐水溶液+2％透明质酸，或者生理盐水溶液+2％透明质酸+配体滴眼，以作为对照，具体为分别采用生理盐水溶液+2％透明质酸，生理盐水溶液+2％透明质酸+10μmol/L双氯芬酸钠，生理盐水溶液+2％透明质酸+5μmol/L维生素A+5％吐温80，生理盐水溶液+2％透明质酸+5μmol/L木犀草苷滴眼。此外，另有1组正常C57BL/6小鼠作为对照(5只)。滴眼2周后，处死动物，剥离视网膜并进行EB染色观察视网膜血管渗漏情况，以及Trypsin酶解观察新生血管密度。
结果如表31所示，表中显示，与正常对照组相比，AMD C57BL/6小鼠不加任何处理的对照组视网膜血管渗漏、新生血管数量均显著提升；而滴加了人源TTR或者人源TTR/双氯芬酸钠、人源TTR/维生素A、人源TTR/木犀草苷组的视网膜渗漏情况及新生血管情况得到显著的缓解。
表31人源TTR/人源TTR-配体分子复合物治疗激光视网膜光凝诱导C57BL/6小鼠AMD病理状况
Figure PCTCN2020128588-appb-000059
Figure PCTCN2020128588-appb-000060
实施例29：人源TTR-配体分子复合物滴眼方式治疗ROP(早产儿视网膜病变)SD大鼠
出生一周乳鼠(SD大鼠)，放入高压氧舱饲养，正常对照组放入正常环境饲养(正常对照组，5只)。高压氧舱饲养5天后取出，获得ROP SD大鼠。将所有ROP SD大鼠连同正常对照组一起在正常环境中继续饲养5天。在该5天中，ROP SD大鼠分为5组，1组为ROP SD小鼠不加任何处理(5只)，另外4组均为5只/组，左眼、右眼各每日滴加1次，每次30μL，其中，左眼为采用实施例5所制得的人源TTR滴眼，或者采用实施例5所制得的人源TTR+配体滴眼，具体为分别采用10μmol/L人源TTR(生理盐水溶液+2％透明质酸)、10μmol/L人源TTR(生理盐水溶液+2％透明质酸+10μmol/L双氯芬酸钠)、10μmol/L人源TTR(生理盐水溶液+2％透明质酸+5μmol/L维生素A+5％吐温80)、10μmol/L人源TTR(生理盐水溶液+2％透明质酸+5μmol/L木犀草苷)滴眼，右眼为采用生理盐水溶液+2％透明质酸，或者生理盐水溶液+2％透明质酸+配体滴眼，以作为对照，具体为分别采用生理盐水溶液+2％透明质酸，生理盐水溶液+2％透明质酸+10μmol/L双氯芬酸钠，生理盐水溶液+2％透明质酸+5μmol/L维生素A+5％吐温80，生理盐水溶液+2％透明质酸+5μmol/L木犀草苷滴眼。正常环境饲养5天后处死乳鼠并剥取视网膜进行EB染色，观察视网膜血管渗漏情况。
结果如表32所示，表中显示，与正常对照组相比，ROP SD大鼠不加任何处理的对照组视网膜血管渗漏显著提升；而滴加了人源TTR或者人源TTR/双氯芬酸钠、人源TTR/维生素A、人源TTR/木犀草苷组的视网膜渗漏情况得到显著的缓解。
表32人源TTR/人源TTR-配体分子复合物治疗高压氧舱诱导SD大鼠乳鼠ROP病理状况
Figure PCTCN2020128588-appb-000061
Figure PCTCN2020128588-appb-000062
结合实施例27-29的数据还可以看出，即使是正常的SD大鼠，成年的SD大鼠的视网膜渗漏情况要比幼年的SD大鼠严重，另外，其他一些原因，例如，实验操作、个体差异等，也可能会造成视网膜渗漏以及新生血管数增加，这就表明即使是未经造模的正常对照眼也存在一定程度的视网膜渗漏以及新生血管增加的情况。通过使用人源TTR/双氯芬酸钠或者人源TTR/维生素A后，不仅可以治疗DR、AMD以及ROP，还可以起到滋养作用，对眼部本身因其他原因产生的视网膜渗漏以及新生血管增加的情况也有一定的缓解效果。
对比例1：人源TTR使用不同质粒进行重组表达
(1)重组质粒pET-28a-ttr,pQE-30-ttr，pQE-60-ttr的构建：成熟TTR优化的DNA序列(如SEQ ID NO:2所示)通过Nde I及Hind III两个酶切位点连接入pET-28a质粒(pET-28a、pQE-30，pQE-60均购于ATCC)，构建了pET-28a-ttr质粒；成熟TTR优化的DNA序列(如SEQ ID NO:2所示)通过BamH I及Hind III两个酶切位点连接入pQE-30质粒，构建了pQE-30-ttr质粒；成熟TTR优化的DNA序列(如SEQ ID NO:2所示)通过EcoR I及Hind III两个酶切位点连接入pQE-60质粒，构建了pQE-60-ttr质粒。经测序验证(南京金斯瑞生物科技有限公司)，构建成功。
(2)重组人源TTR的表达和纯化：将步骤(1)构建的pET-28a-ttr，pQE-30-ttr，pQE-60-ttr质粒以及实施例5中所构建的pETx-rhaPBAD-ttr转化入E.coli BL21(DE3)细胞，将所得重组E.coli BL21(DE3)在LB培养基中培养，制备种子液，再以5％的接种量，接入至5L TB培养基中，温度37℃，搅拌桨转速150rpm，培养至OD 600为1.5-2.0；前三种培养基加入0.2mM IPTG诱导18h，最后一种培养基加入1.6％鼠李糖诱导 18h。通过高压均质机将菌体破碎后，上清液通过Ni+柱层析制备得到人源TTR。使用内毒素吸附柱(Pierce TM High Capacity Endotoxin Removal Spin Columns,ThermoFisher)去除内毒素，通过0.22μm孔径滤膜去除残留菌体。所得人源TTR蛋白产量如表33所示。
表33人源TTR使用不同质粒进行重组表达
Figure PCTCN2020128588-appb-000063
由以上结果可知，使用pET-28a、pQE-30，pQE-60等进行表达时，均不存在可溶性表达。
对比例2：
相对于实施例7中的表7-1，滴加TTR的浓度为1μmol/L时，玻璃体及眼底内人源TTR的浓度仅为0.10±0.00μmol/L。
对比例3：
具体实施方式同实施例9，区别在于，按照实施例2的方法表达不与TTR融合的GFP蛋白(Genbank登录号为QAA95705.1)，将未融合TTR的GFP蛋白按照实施例9相同的操作步骤对SD大鼠及新西兰大耳兔进行滴眼测试，结果显示，GFP未进入SD大鼠及新西兰大耳兔玻璃体内(表34，35)。
表34每次滴加GFP含量0.6nmol，滴加不同次数后GFP到达SD大鼠眼内的效果
Figure PCTCN2020128588-appb-000064
其中，“-”表示未检出(下同)。
表35每次滴加GFP含量0.6nmol，滴加不同次数后GFP到达新西兰大耳兔眼内的效果
Figure PCTCN2020128588-appb-000065
对比例4：
具体实施方式同实施例10，区别在于，按照实施例3的方法表达不与TTR融合的Lysozyme蛋白(Genbank登录号为AAL69327.1)，将未融合的Lysozyme蛋白按照实施例10相同的操作步骤对SD大鼠及新西兰大耳兔进行滴眼测试，结果显示，Lysozyme未进入SD大鼠及新西兰大耳兔玻璃体内(表36，37)。
表36每次滴加Lysozyme含量0.6nmol，滴加不同次数后Lysozyme到达SD大鼠眼内的效果
Figure PCTCN2020128588-appb-000066
表37每次滴加Lysozyme含量0.6nmol滴加不同次数后不同处理方式下Lysozyme到达新西兰大耳兔眼内的效果
Figure PCTCN2020128588-appb-000067
对比例5：
具体实施方式同实施例11，区别在于，按照实施例4的方法表达不与TTR融合的Ovalbumin蛋白(UniProt登录号为P01012)，将未融合的Ovalbumin蛋白按照实施例11相同的操作步骤对SD大鼠及新西兰大耳兔进行滴眼测试，结果显示，Ovalbumin未进入SD大鼠及新西兰大耳兔玻璃体内(表38，39)。
表38每次滴加Ovalbumin含量0.6nmol，每日滴加不同次数后Ovalbumin到达SD 大鼠眼内的效果
Figure PCTCN2020128588-appb-000068
表39每次滴加Ovalbumin含量0.6nmol，每日滴加不同次数后Ovalbumin到达新西兰大耳兔眼内的效果
Figure PCTCN2020128588-appb-000069
对比例6：
(1)将实施例5制得的人源TTR配置为5-30μmol/L(含有生理盐水)，分别对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼，经过3h后处死，取玻璃体与眼底样本提取蛋白，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过ELISA，测定C57BL/6小鼠及SD大鼠的玻璃体与眼底样本中人源TTR的含量。
(2)将实施例5制得的人源TTR配置为10μmol/L(含有生理盐水及0-8mg/mL PEG400(购于国药，分子量360-440))，分别对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼，经过3-72h后处死，取玻璃体与眼底样本提取蛋白，使用兔抗-His-tag抗体为一抗，驴抗兔抗体为二抗，通过ELISA，测定C57BL/6小鼠及SD大鼠的玻璃体与眼底样本中人源TTR的含量。
结果显示，滴眼3h后玻璃体与眼底样本中人源TTR含量达到最高峰；滴眼液中，添加了PEG400之后，玻璃体与眼底样本中人源TTR含量提高了20％以上，且当PEG400含量为6mg/mL时，滴眼3h后玻璃体与眼底样本中人源TTR含量达到最高峰；滴眼后，人源TTR在C57BL/6小鼠及SD大鼠玻璃体与眼底样本内的含量半衰期接近60h，说明可以在玻璃体与眼底样本内有效存在60h，有足够的治疗浓度和治疗时间(表40)。
结合实施例21和22中的结果可知，虽然PEG400可有效促进TTR在玻璃体和眼底 样本中的含量，但将其用于治疗患有DR或AMD的老鼠时，治疗效果相对较差。这就表明，TTR透过量的增加，并不意味着治疗效果增加，TTR、TTR合适的透过量以及合适的辅料，相互牵制，协同配合，形成一个有机整体，最终才能获得一个较佳的治疗效果。
此外，从表25和表27可知，即使是正常的SD大鼠，成年的SD大鼠的视网膜渗漏情况要比幼年的SD大鼠严重，另外，其他一些原因，例如，实验操作、个体差异等，也可能会造成视网膜渗漏以及新生血管数增加，这就表明即使是未经造模的正常对照眼也存在一定程度的视网膜渗漏以及新生血管增加的情况。通过将TTR和硫酸软骨素A钠盐配合使用后，不仅可以治疗DR、AMD以及ROP，还可以起到滋养作用，对眼部本身因其他原因产生的视网膜渗漏以及新生血管增加的情况也有一定的缓解效果。
表40人源TTR跨越C57BL/6小鼠及SD大鼠角膜屏障进入玻璃体及眼底
Figure PCTCN2020128588-appb-000070
Figure PCTCN2020128588-appb-000071
对比例7：
具体实施方式同实施例26，区别在于，将未与TTR结合的各个配体分子按照实施例26相同的操作步骤对C57BL/6小鼠(8周龄)及SD大鼠(8周龄)进行滴眼测试，结果显示，各个配体分子单独无法进入C57BL/6小鼠及SD大鼠的玻璃体及眼底(表41)。
表41各个配体分子跨越C57BL/6小鼠及SD大鼠角膜屏障进入玻璃体及眼底
Figure PCTCN2020128588-appb-000072
Figure PCTCN2020128588-appb-000073
对比例8：
具体实施方式同实施例27，表42中显示了其中右眼滴眼后的结果数据。结果显示，各个配体分子单独滴眼无法改善STZ诱导SD大鼠DR病理状况(表42)。
表42各配体单独滴眼治疗STZ诱导SD大鼠DR病理状况
Figure PCTCN2020128588-appb-000074
Figure PCTCN2020128588-appb-000075
对比例9：
具体实施方式同实施例28，表43中显示了其中右眼滴眼后的结果数据。结果显示，各个配体分子单独滴眼无法改善激光视网膜光凝诱导C57BL/6小鼠AMD病理状况(表43)。
表43各配体单独滴眼治疗激光视网膜光凝诱导C57BL/6小鼠AMD病理状况
Figure PCTCN2020128588-appb-000076
对比例10：
具体实施方式同实施例29，表44中显示了其中右眼滴眼后的结果数据。结果显示，各个配体分子单独滴眼无法改善高压氧舱诱导SD大鼠乳鼠ROP病理状况(表44)。
表44各配体单独滴眼治疗高压氧舱诱导SD大鼠乳鼠ROP病理状况
Figure PCTCN2020128588-appb-000077
Figure PCTCN2020128588-appb-000078
对比例11：
磺胺甲恶唑(sulfamethoxazole)的抗菌谱广，抗菌作用强，能阻碍细菌生长，对葡萄球菌、大肠杆菌特别有效；适用于呼吸系统、泌尿系统及肠道感染等；主要用于治疗禽霍乱等。其可用于制备滴眼液例如复方磺胺甲恶唑钠滴眼液(本品为复方制剂，每10毫升含磺胺甲恶唑钠400毫克，氨基己酸200毫克、甘草酸二钾10毫克、马来酸氯苯那敏2毫克。主要用于敏感菌所引起的细菌性结膜炎、睑腺炎(麦粒肿)及细菌性眼睑炎)。
通过Discovery studio软件进行分子模拟发现磺胺甲恶唑能够与TTR多聚体稳定结合。图17A中显示的蛋白结构为TTR二聚体，磺胺甲恶唑配体分子使用箭头指示，一分子TTR二聚体可以结合一分子磺胺甲恶唑。图17B中显示了磺胺甲恶唑与TTR氨基酸残基的相互作用。
参考实施例25的步骤，在10μmol/L的TTR溶液(1000μL)中，以1μL/min的速度滴加100μmol/L的磺胺甲恶唑钠盐溶液，使用内置软件计算亲和结合平衡解离常数Kd为7.03×10 -8mol/L。
参照实施例26的步骤使用TTR/磺胺甲恶唑钠盐对大鼠和小鼠进行滴眼3-72小时后，大鼠和小鼠的角膜出现损伤，即，磺胺甲恶唑钠盐与TTR配合使用时会烧伤角膜，不具有生物安全性。可见，用Discovery studio软件分子模拟筛选出同样具有生物活性、具有消炎作用且均可以与TTR结合的配体后，进行试验验证时并不是都可以提高治疗效果。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (15)

  1. 转甲状腺素蛋白作为蛋白质类和/或多肽类药物通过眼部屏障进入眼内的载体方面的应用，所述转甲状腺素蛋白如(a)、(b)或(c)所示：
    (a)由SEQ ID NO:1所示氨基酸组成的蛋白质；
    (b)在(a)中的氨基酸序列上经过取代、缺失或添加一个或几个氨基酸的序列所示的具有抑制血管新生功能的由(a)衍生的蛋白质；
    (c)在(a)或(b)中的氨基酸序列上经过亲水性修饰或疏水性修饰的序列所示的蛋白质。
  2. 如权利要求1所述的应用，其特征在于，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过22个氨基酸的取代、25个氨基酸的取代、或5个氨基酸的缺失；
    和/或，所述(c)中，所述亲水性修饰或疏水性修饰在(a)中的氨基酸序列上的第10位的半胱氨酸上进行；
    较佳地：
    所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列的T3、T5、I26、N27、H31、R34、A36、A37、D38、D39、T40、S50、E61、E63、V65、I68、K70、I73、A81、H90、E92、P102、R104、T123、K126和/或E127发生取代；或在(a)中的氨基酸序列的第123-127位发生缺失；优选在(a)中的氨基酸序列上经过T3G、T5A、I26V、N27D、H31K、R34K、A36T、D39G、T40S、S50A、E61D、E63K、V65T、I68V、K70R、I73L、H90Y、P102H、R104H、T123S、K126Q和E127N的取代，或在(a)中的氨基酸序列上经过T3G、T5A、I26V、N27D、H31K、R34K、A36T、A37S、D38E、D39G、T40S、S50A、E61D、E63K、I68V、K70R、I73L、A81T、H90F、E92D、P102H、R104H、T123S、K126Q和E127N的取代；
    和/或，所述(c)中，所述疏水性修饰为在(a)中的氨基酸序列上的第10位的半胱氨酸上使用长链疏水片段例如正十二烷进行修饰、或、在(a)中的氨基酸序列上的第10位的半胱氨酸上通过马来酰亚胺连接正十二烷进行修饰；
    更佳地：
    所述(b)中，所述的由(a)衍生的蛋白质的氨基酸序列如SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11所示。
  3. 如权利要求1或2所述的应用，其特征在于，所述转甲状腺素蛋白与所述蛋白质 类和/或多肽类药物融合表达；所述融合优选是将所述蛋白质类和/或多肽类药物融合在所述转甲状腺素蛋白的N端或C端；所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合优选是在微生物细胞中表达，并经过纯化；所述纯化优选是通过内毒素吸附柱去除内毒素，再通过0.22μm孔径滤膜去除残留菌体；
    和/或，所述蛋白质类和/或多肽类药物包括溶菌酶、白蛋白和/或EGFR抗体，且分子量不超过45kDa；所述溶菌酶优选为鸡蛋清溶菌酶，其GenBank登录号为AAL69327.1；所述白蛋白优选为鸡蛋清白蛋白；
    和/或，所述蛋白质类和/或多肽类药物包括治疗眼部视网膜渗漏和/或视网膜血管新生类眼部疾病例如糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的蛋白质类和/或多肽类药物。
  4. 如权利要求1～3任一项所述的应用，其特征在于，编码所述转甲状腺素蛋白的核苷酸序列如SEQ ID NO:2所示；
    和/或，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架质粒中的启动子为鼠李唐诱导型启动子，优选为rhaPBAD启动子；
    和/或，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架载体为pET-21a或与其具有25％及以上同源性的载体，所述与其具有25％及以上同源性的载体的序列优选如SEQ ID NO:8所示；
    和/或，表达所述转甲状腺素蛋白的重组质粒的核苷酸序列如SEQ ID NO:3所示；
    和/或，所述转甲状腺素蛋白是在微生物细胞中表达，并优选经过纯化；所述的微生物细胞优选为大肠杆菌，所述大肠杆菌优选包括E.coli BL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α、E.coli K12或E.coli TOP10；所述纯化优选是通过内毒素吸附柱去除内毒素，通过0.22μm孔径滤膜去除残留菌体；
    和/或，表达所述转甲状腺素蛋白时，通过培养包含所述转甲状腺素蛋白的基因的转化体至所得菌体的OD 600达到1.5-2.0，例如1.6、1.7、1.8或1.9时进行表达；
    和/或，表达所述转甲状腺素蛋白时为使用诱导表达的试剂进行诱导表达，所述诱导表达的试剂的质量体积百分比为0.1-2％，例如0.2％、0.3％、0.4％、0.5％、0.7％、0.8％、1.2％或1.6％，所述诱导表达的时间优选为8-20h，例如10h、12h、14h、16h、17h、18h或19h；所述诱导表达的试剂优选为鼠李糖或者IPTG。
  5. 转甲状腺素蛋白和/或转甲状腺素蛋白与药物组成的融合蛋白在制备滴剂中的应用，所述药物为蛋白质类和/或多肽类药物，所述转甲状腺素蛋白如(a)、(b)或(c)所示：
    (a)由SEQ ID NO:1所示氨基酸组成的蛋白质；
    (b)在(a)中的氨基酸序列上经过取代、缺失或添加一个或几个氨基酸的序列所示的具有抑制血管新生功能的由(a)衍生的蛋白质；
    (c)在(a)或(b)中的氨基酸序列上经过亲水性修饰或疏水性修饰的序列所示的蛋白质。
  6. 如权利要求5所述的应用，其特征在于，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过22个氨基酸的取代、25个氨基酸的取代、或5个氨基酸的缺失；
    和/或，所述(c)中，所述亲水性修饰或疏水性修饰在(a)中的氨基酸序列上的第10位的半胱氨酸上进行；
    较佳地：
    所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列的T3、T5、I26、N27、H31、R34、A36、A37、D38、D39、T40、S50、E61、E63、V65、I68、K70、I73、A81、H90、E92、P102、R104、T123、K126和/或E127发生取代；或在(a)中的氨基酸序列的第123-127位发生缺失；优选在(a)中的氨基酸序列上经过T3G、T5A、I26V、N27D、H31K、R34K、A36T、D39G、T40S、S50A、E61D、E63K、V65T、I68V、K70R、I73L、H90Y、P102H、R104H、T123S、K126Q和E127N的取代，或在(a)中的氨基酸序列上经过T3G、T5A、I26V、N27D、H31K、R34K、A36T、A37S、D38E、D39G、T40S、S50A、E61D、E63K、I68V、K70R、I73L、A81T、H90F、E92D、P102H、R104H、T123S、K126Q和E127N的取代；
    和/或，所述(c)中，所述疏水性修饰为在(a)中的氨基酸序列上的第10位的半胱氨酸上使用长链疏水片段例如正十二烷进行修饰、或、在(a)中的氨基酸序列上的第10位的半胱氨酸上通过使用马来酰亚胺连接正十二烷进行修饰；
    更佳地：
    所述(b)中，所述的由(a)衍生的蛋白质的氨基酸序列如SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11所示。
  7. 如权利要求5或6所述的应用，其特征在于，当所述滴剂含有转甲状腺素蛋白与药物组成的融合蛋白时，所述融合蛋白的含量4-30μmol/L，优选10-15μmol/L；
    和/或，当所述滴剂含有转甲状腺素蛋白时，所述转甲状腺素蛋白的含量为4-30μmol/L，优选5-30μmol/L，更优选10-20μmol/L，例如10、15、20μmol/L；
    和/或，所述滴剂中还含有生理盐水；
    和/或，所述滴剂中还含有表面活性剂，所述表面活性剂例如为吐温80，其含量优选为5v/v％；
    和/或，所述滴剂中还含有药学上可接受的辅料，所述药学上可接受的辅料选自羧甲基纤维素或其盐、硫酸软骨素或其盐、右旋糖酐、和、透明质酸中的一种或多种；其中，所述羧甲基纤维素或其盐的黏度优选为800-1200CP；和/或，所述硫酸软骨素优选为硫酸软骨素A；和/或，所述右旋糖酐优选为右旋糖酐70；和/或，所述的“羧甲基纤维素或其盐”、“硫酸软骨素或其盐”中的“盐”独立地优选为钠盐或钙盐，例如为羧甲基纤维素钠或硫酸软骨素A钠盐；和/或，所述透明质酸的分子量优选为10000-500000；和/或，所述羧甲基纤维素或其盐的浓度优选为0-8mg/mL但不为0，更优选为2、4、6或8mg/mL；和/或，所述硫酸软骨素或其盐的浓度优选为0-40mg/mL但不为0，更优选为10、20、30或40mg/mL；和/或，所述右旋糖酐的浓度优选为0-0.8mg/mL但不为0，更优选为0.2、0.4、0.6或0.8mg/mL；和/或，所述透明质酸的含量优选≤6％，优选1-4％，更优选2％；
    和/或，所述滴剂中还含有化合物、其药学上可接受的盐或其糖苷；所述化合物选自双氯芬酸、维生素A和木犀草素中的一种或多种；其中，所述的药学上可接受的盐优选为钠盐，例如为双氯芬酸钠；和/或，所述的糖苷优选为木犀草苷；和/或，所述的维生素A优选为维生素A1和/或维生素A2；和/或，所述双氯芬酸或其盐的含量优选为5-20μmol/L，例如为10μmol/L；和/或，所述维生素A的含量优选为2-10μmol/L，例如为5μmol/L；和/或，所述木犀草素或其糖苷的含量优选为2-10μmol/L，例如为5μmol/L；
    和/或，所述滴剂优选滴眼液；
    和/或，所述滴剂为抑制眼部视网膜渗漏和/或降低视网膜新生血管数的滴剂，优选为治疗糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的滴剂；
    和/或，所述滴剂以每日滴加1-3次、每次滴加量优选为0.3-0.8nmol蛋白/眼进行施用；
    和/或，所述滴剂以每日2次、每次1滴、持续3个月进行施用；和/或，所述滴剂以每日1次、每次1滴、持续5天进行施用；和/或，所述滴剂以每日2次、每次1滴、持续2周进行施用；
    和/或，编码所述转甲状腺素蛋白的核苷酸序列如SEQ ID NO:2所示；
    和/或，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架质粒中的启动子为鼠李唐诱导型启动子，优选为rhaPBAD启动子；
    和/或，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架载体为pET-21a或与其具有25％及以上同源性的载体，所述与其具有25％及以上同 源性的载体的序列优选如SEQ ID NO:8所示；
    和/或，表达所述转甲状腺素蛋白的重组质粒的核苷酸序列如SEQ ID NO:3所示；
    和/或，所述转甲状腺素蛋白是在微生物细胞中表达，并优选经过纯化；所述的微生物细胞优选为大肠杆菌，所述大肠杆菌优选包括E.coli BL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α、E.coli K12或E.coli TOP10；所述纯化优选是通过内毒素吸附柱去除内毒素，通过0.22μm孔径滤膜去除残留菌体；
    和/或，表达所述转甲状腺素蛋白时，通过培养包含所述转甲状腺素蛋白的基因的转化体至所得菌体的OD 600达到1.5-2.0，例如1.6、1.7、1.8或1.9时进行表达；
    和/或，表达所述转甲状腺素蛋白时为使用诱导表达的试剂进行诱导表达，所述诱导表达的试剂的质量体积百分比为0.1-2％，例如0.2％、0.3％、0.4％、0.5％、0.7％、0.8％、1.2％或1.6％，所述诱导表达的时间优选为8-20h，例如10h、12h、14h、16h、17h、18h或19h；所述诱导表达的试剂优选为鼠李糖或者IPTG；
    和/或，所述融合蛋白的序列如SEQ ID NO:6或SEQ ID NO:7所示；
    和/或，所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合表达；所述融合优选是将所述蛋白质类和/或多肽类药物融合在所述转甲状腺素蛋白的N端或C端；所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合优选是在微生物细胞中表达，并经过纯化；所述纯化优选是通过内毒素吸附柱去除内毒素，再通过0.22μm孔径滤膜去除残留菌体；
    和/或，所述蛋白质类和/或多肽类药物包括溶菌酶、白蛋白和/或EGFR抗体，且分子量不超过45kDa；所述溶菌酶优选为鸡蛋清溶菌酶，其GenBank登录号为AAL69327.1；所述白蛋白优选为鸡蛋清白蛋白；
    和/或，所述蛋白质类和/或多肽类药物包括治疗眼部视网膜渗漏和/或视网膜血管新生类眼部疾病例如糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的蛋白质类和/或多肽类药物。
  8. 一种滴剂，其特征在于，其含有转甲状腺素蛋白和/或转甲状腺素蛋白与药物组成的融合蛋白；所述药物为蛋白质类和/或多肽类药物，所述转甲状腺素蛋白如(a)、(b)或(c)所示：
    (a)由SEQ ID NO:1所示氨基酸组成的蛋白质；
    (b)在(a)中的氨基酸序列上经过取代、缺失或添加一个或几个氨基酸的序列所示的具有抑制血管新生功能的由(a)衍生的蛋白质；
    (c)在(a)或(b)中的氨基酸序列上经过亲水性修饰或疏水性修饰的序列所示的 蛋白质。
  9. 如权利要求8所述的滴剂，其特征在于，所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列上经过22个氨基酸的取代、25个氨基酸的取代、或5个氨基酸的缺失；
    和/或，所述(c)中，所述亲水性修饰或疏水性修饰在(a)中的氨基酸序列上的第10位的半胱氨酸上进行；
    较佳地：
    所述(b)中，所述的由(a)衍生的蛋白质为在(a)中的氨基酸序列的T3、T5、I26、N27、H31、R34、A36、A37、D38、D39、T40、S50、E61、E63、V65、I68、K70、I73、A81、H90、E92、P102、R104、T123、K126和/或E127发生取代；或在(a)中的氨基酸序列的第123-127位发生缺失；优选在(a)中的氨基酸序列上经过T3G、T5A、I26V、N27D、H31K、R34K、A36T、D39G、T40S、S50A、E61D、E63K、V65T、I68V、K70R、I73L、H90Y、P102H、R104H、T123S、K126Q和E127N的取代，或在(a)中的氨基酸序列上经过T3G、T5A、I26V、N27D、H31K、R34K、A36T、A37S、D38E、D39G、T40S、S50A、E61D、E63K、I68V、K70R、I73L、A81T、H90F、E92D、P102H、R104H、T123S、K126Q和E127N的取代；
    和/或，所述(c)中，所述疏水性修饰为在(a)中的氨基酸序列上的第10位的半胱氨酸上使用长链疏水片段例如正十二烷进行修饰、或、在(a)中的氨基酸序列上的第10位的半胱氨酸上通过使用马来酰亚胺连接正十二烷进行修饰；
    更佳地：
    所述(b)中，所述的由(a)衍生的蛋白质的氨基酸序列如SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11所示。
  10. 如权利要求8或9所述的滴剂，其特征在于，当所述滴剂含有转甲状腺素蛋白与药物组成的融合蛋白时，所述融合蛋白的含量4-30μmol/L，优选10-15μmol/L；
    和/或，当所述滴剂含有转甲状腺素蛋白时，所述转甲状腺素蛋白的含量为4-30μmol/L，优选5-30μmol/L，更优选10-20μmol/L，例如10、15、20μmol/L；
    和/或，所述滴剂中还含有生理盐水；
    和/或，所述滴剂中还含有表面活性剂，所述表面活性剂例如为吐温80，其含量优选为5v/v％；
    和/或，所述滴剂优选滴眼液；
    和/或，所述滴剂为抑制眼部视网膜渗漏和/或降低视网膜新生血管数的滴剂，优选为 治疗糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的滴剂；
    和/或，所述滴剂以每日滴加1-3次、每次滴加量优选为0.3-0.8nmol蛋白/眼进行施用；
    和/或，所述滴剂以每日2次、每次1滴、持续3个月进行施用；和/或，所述滴剂以每日1次、每次1滴、持续5天进行施用；和/或，所述滴剂以每日2次、每次1滴、持续2周进行施用；
    和/或，编码所述转甲状腺素蛋白的核苷酸序列如SEQ ID NO:2所示；
    和/或，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架质粒中的启动子为鼠李唐诱导型启动子，优选为rhaPBAD启动子；
    和/或，所述转甲状腺素蛋白通过使用重组表达载体进行表达，所述重组表达载体的骨架载体为pET-21a或与其具有25％及以上同源性的载体，所述与其具有25％及以上同源性的载体的序列优选如SEQ ID NO:8所示；
    和/或，表达所述转甲状腺素蛋白的重组质粒的核苷酸序列如SEQ ID NO:3所示；
    和/或，所述转甲状腺素蛋白是在微生物细胞中表达，并优选经过纯化；所述的微生物细胞优选为大肠杆菌，所述大肠杆菌优选包括E.coli BL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α、E.coli K12或E.coli TOP10；所述纯化优选是通过内毒素吸附柱去除内毒素，通过0.22μm孔径滤膜去除残留菌体；
    和/或，表达所述转甲状腺素蛋白时，通过培养包含所述转甲状腺素蛋白的基因的转化体至所得菌体的OD 600达到1.5-2.0，例如1.6、1.7、1.8或1.9时进行表达；
    和/或，表达所述转甲状腺素蛋白时为使用诱导表达的试剂进行诱导表达，所述诱导表达的试剂的质量体积百分比为0.1-2％，例如0.2％、0.3％、0.4％、0.5％、0.7％、0.8％、1.2％或1.6％，所述诱导表达的时间优选为8-20h，例如10h、12h、14h、16h、17h、18h或19h；所述诱导表达的试剂优选为鼠李糖或者IPTG；
    和/或，所述融合蛋白的序列如SEQ ID NO:6或SEQ ID NO:7所示；
    和/或，所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合表达；所述融合优选是将所述蛋白质类和/或多肽类药物融合在所述转甲状腺素蛋白的N端或C端；所述转甲状腺素蛋白与所述蛋白质类和/或多肽类药物融合优选是在微生物细胞中表达，并经过纯化；所述纯化优选是通过内毒素吸附柱去除内毒素，再通过0.22μm孔径滤膜去除残留菌体；
    和/或，所述蛋白质类和/或多肽类药物包括溶菌酶、白蛋白和/或EGFR抗体，且分子量不超过45kDa；所述溶菌酶优选为鸡蛋清溶菌酶，其GenBank登录号为AAL69327.1； 所述白蛋白优选为鸡蛋清白蛋白；
    和/或，所述蛋白质类和/或多肽类药物包括治疗眼部视网膜渗漏和/或视网膜血管新生类眼部疾病例如糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的蛋白质类和/或多肽类药物。
  11. 如权利要求8-10任一项所述的滴剂，其特征在于，所述滴剂中还含有药学上可接受的辅料，所述药学上可接受的辅料选自羧甲基纤维素或其盐、硫酸软骨素或其盐、右旋糖酐、和、透明质酸中的一种或多种；
    较佳地：
    所述羧甲基纤维素或其盐的黏度为800-1200CP；
    和/或，所述硫酸软骨素为硫酸软骨素A；
    和/或，所述右旋糖酐为右旋糖酐70；
    和/或，所述的“羧甲基纤维素或其盐”、“硫酸软骨素或其盐”中的“盐”独立地为钠盐或钙盐，例如为羧甲基纤维素钠或硫酸软骨素A钠盐；
    和/或，所述透明质酸的分子量为10000-500000；
    和/或，所述羧甲基纤维素或其盐的浓度为0-8mg/mL但不为0，优选为2、4、6或8mg/mL；
    和/或，所述硫酸软骨素或其盐的浓度为0-40mg/mL但不为0，优选为10、20、30或40mg/mL；
    和/或，所述右旋糖酐的浓度为0-0.8mg/mL但不为0，优选为0.2、0.4、0.6或0.8mg/mL；
    和/或，所述透明质酸的含量≤6％，优选1-4％，更优选2％。
  12. 如权利要求8-11任一项所述的滴剂，其特征在于，所述滴剂中还含有化合物、其药学上可接受的盐或其糖苷；所述化合物选自双氯芬酸、维生素A和木犀草素中的一种或多种；
    较佳地：
    所述的药学上可接受的盐为钠盐，例如为双氯芬酸钠；
    和/或，所述的糖苷为木犀草苷；
    和/或，所述的维生素A为维生素A1和/或维生素A2；
    和/或，所述双氯芬酸或其盐的含量为5-20μmol/L，例如为10μmol/L；
    和/或，所述维生素A的含量为2-10μmol/L，例如为5μmol/L；
    和/或，所述木犀草素或其糖苷的含量为2-10μmol/L，例如为5μmol/L。
  13. 如权利要求8-12任一项所述的滴剂在制备抑制眼部视网膜渗漏和/或降低视网膜新生血管数的药物中的应用；优选为在制备治疗糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变的药物中的应用。
  14. 如权利要求8-12任一项所述的滴剂在治疗眼部疾病中的应用；所述的眼部疾病优选为眼部视网膜渗漏和/或视网膜血管新生相关眼部疾病，更优选为糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变。
  15. 一种治疗眼部视网膜渗漏和/或视网膜血管新生类眼部疾病例如糖尿病视网膜病变、老年性黄斑变性和/或早产儿视网膜病变等的方法，包括向需要治疗的患者施用包含如权利要求8-12任一项所述的滴剂。
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