CN112043820B - Eye preparation and preparation method and application thereof - Google Patents

Abstract

The present invention provides an ophthalmic preparation comprising a compound, a pharmaceutically acceptable salt thereof or a glycoside thereof, and transthyretin; the compound is selected from one or more of diclofenac, vitamin A and luteolin. The invention also provides a preparation method and application of the ophthalmic preparation. The transthyretin and the compound in the ophthalmic preparation can be cooperatively matched, so that the treatment effect on the ocular diseases related to ocular angiogenesis, ocular retinal leakage and the like can be remarkably improved when the ophthalmic preparation is used for treating the ocular diseases.

Description

Eye preparation and preparation method and application thereof

Technical Field

The invention belongs to the technical field of protein engineering, and particularly relates to an ophthalmic preparation containing transthyretin and a specific compound, and a preparation method and application thereof.

Background

Diclofenac and its sodium salt: diclofenac and its sodium salt are non-steroidal anti-inflammatory drugs, and have obvious analgesic, anti-inflammatory and antipyretic effects. Diclofenac sodium eye drops for treating uveitis, keratitis and scleritis, and inhibiting formation of corneal neovascularization; treating inflammatory reactions after intraocular surgery, laser filter screen formation or various eye injuries; the miosis reaction in cataract surgery is inhibited; used for relieving pain and diminishing inflammation after excimer laser keratotomy; treating allergic eye diseases such as vernal conjunctivitis and seasonal allergic conjunctivitis; preventing and treating cataract, artificial lens postoperative inflammation and macular cystoid edema; and promoting formation of filtering bleb after glaucoma filtration. The diclofenac sodium eye drops are used for the ocular surface or the open wound of the eye, and the current commercial product is Difei, and the content is 1 mg/mL. The structural formulas of diclofenac and diclofenac sodium are shown as follows:

diclofenac acid, which is a compound of diclofenac,

diclofenac sodiumSodium salt.

Vitamin A: vitamin A includes retinol (A1) and 3-dehydroretinol (A2), and is a kind of fat-soluble vitamin, which is stable to heat, acid and alkali. Vitamin A has multiple physiological functions of promoting growth and reproduction, and maintaining normal secretion of bone, epithelial tissue, vision and mucous epithelium, etc., and vitamin A and its analogs have the effect of preventing precancerous lesion. Vitamin a deficiency manifests as delayed growth, diminished dark adaptation and nyctalopia. The vitamin A eye drops are used for promoting the growth of corneal cells and treating xerophthalmia. Wherein the structural formulas of the retinol (A1) and the 3-dehydroretinol (A2) are shown as follows:

A1，

A2。

luteolin: is a natural flavonoid compound and exists in various plants. Has various pharmacological activities such as anti-inflammation, anti-allergy, uric acid reduction, anti-tumor, antibacterial and antivirus, and is mainly used for relieving cough, eliminating phlegm, diminishing inflammation, reducing uric acid, treating cardiovascular diseases, treating amyotrophic lateral sclerosis, SARS, hepatitis and the like in clinic. Luteolin, in many plants, is present in the form of glycosides. The structural formulas of luteolin and its glycoside forms are respectively as follows:

the content of luteolin in the raw materials of the medicine,

luteoloside.

There is no report of the dynamic specific binding of these drugs To Transthyretin (TTR).

CN109481668A discloses transthyretin (TTR) can be used for preparing medicines for inhibiting angiogenesis, but the effect of the transthyretin for treating eye diseases still needs to be improved.

Disclosure of Invention

Aiming at the defects that the effect is not ideal enough when The Transthyretin (TTR) is used for treating eye diseases alone in the prior art, and the like, the invention provides an eye preparation and a preparation method and application thereof. The transthyretin in the ophthalmic preparation of the present invention can be synergistically combined with a specific compound, so that the therapeutic effect on ocular diseases associated with ocular angiogenesis and/or ocular retinal leakage, etc. can be significantly improved when the ophthalmic preparation is used for treating ocular diseases.

Through a large number of experiments, the inventor unexpectedly finds that when the transthyretin and the specific compound disclosed by the invention are applied to eyes together, the compound can cooperate and act together with the transthyretin, so that eye diseases related to ocular angiogenesis and/or ocular retinal leakage and the like can be effectively treated or relieved.

In order to solve the above technical problems, the present invention provides, in a first aspect, an ophthalmic preparation comprising a compound, a pharmaceutically acceptable salt thereof, a glycoside thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystalline form thereof, and transthyretin; the compound is selected from one or more of diclofenac, vitamin A and luteolin.

In the present invention, the salts are generally pharmaceutically acceptable salts, which generally refer to salts prepared from the compounds of the present invention with relatively nontoxic, pharmaceutically acceptable acids or bases. When compounds of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the prototype of such compounds with a sufficient amount of a pharmaceutically acceptable base, in neat solution or in 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. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a prototype of such a compound with a sufficient amount of a pharmaceutically acceptable acid in neat solution or in a suitable inert solvent. The pharmaceutically acceptable acids include inorganic acids including, but 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, acidic citric acid, oleic acid, tannic acid, pantothenic acid, bitartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, saccharic acid, formic acid, ethanesulfonic acid, pamoic acid (i.e., 4' -methylene-bis (3-hydroxy-2-naphthoic acid)), amino acids (e.g., glutamic acid, arginine), and the like. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they may be converted to base addition salts or acid addition salts. See in particular Berge et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science 66:1-19(1977), or, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P.Heinrich Stahl and Camile G.Wermuth, ed., Wiley-VCH, 2002). In a preferred embodiment, the pharmaceutically acceptable salt is a sodium salt, such as diclofenac sodium.

Preferably, the glycoside is luteolin.

Preferably, the vitamin A is vitamin A1 and/or vitamin A2. In a preferred embodiment of the present invention, the vitamin A is commercially available from national medicine under the code CATOCCHM700908, CAS: 68-26-8.

Preferably, the amount of transthyretin in the ophthalmic formulation is 4-30. mu. mol/L, preferably 5-30. mu. mol/L, more preferably 10-20. mu. mol/L, e.g.10, 15 or 20. mu. mol/L.

Preferably, the amount of diclofenac (or e.g. its sodium salt, diclofenac sodium) in the ophthalmic formulation is 5-20 μmol/L, e.g. 10 μmol/L.

Preferably, the amount of vitamin A in the ophthalmic formulation is 2-10. mu. mol/L, for example 5. mu. mol/L.

Preferably, the ophthalmic formulation has a luteolin (or e.g. luteolin in its glycoside form) content of 2-10 μmol/L, e.g. 5 μmol/L.

Preferably, in the ophthalmic preparation, the transthyretin is as shown in (a), (b) or (c):

(a) the nucleotide sequence of the protein consisting of the amino acid shown in SEQ ID NO.1 is preferably shown in SEQ ID NO. 2;

(b) a protein derived from (a) and having an angiogenesis inhibiting function by substituting, deleting or adding one or more amino acids in the amino acid sequence of (a);

(c) a protein represented by a sequence modified in a hydrophilic or hydrophobic manner in the amino acid sequence in (a) or (b).

Wherein, in the step (b), the amino acid sequence of the protein derived from the step (a) can be shown as SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO. 7.

Wherein in (c) the hydrophilic or hydrophobic modification is performed on the cysteine at position 10 on the amino acid sequence in (a), preferably on the cysteine at position 10 on the amino acid sequence in (a) using a long-chain hydrophobic fragment such as n-dodecane, or on the cysteine at position 10 on the amino acid sequence in (a) by linking n-dodecane using maleimide.

It will be appreciated by those skilled in the art that the co-ordination and results of The Transthyretin (TTR) with the compounds of the present invention to enhance therapeutic effect should be equally applicable to the derivative proteins of TTR mentioned in the prior art (e.g., CN111437398A, which is incorporated herein by reference in its entirety) (e.g., rat-derived TTR with the amino acid sequence shown in SEQ ID NO:5, mouse-derived TTR with the amino acid sequence shown in SEQ ID NO:6, or human-derived TTR-CL with the amino acid sequence shown in SEQ ID NO:7, which is the protein product after C-TNPKE loss of human-derived mature TTR).

Preferably, the ophthalmic preparation further comprises pharmaceutically acceptable auxiliary materials such as physiological saline, a surfactant and/or hyaluronic acid, wherein the surfactant is tween 80, and the content of the surfactant is preferably 5% (v/v); the content of the hyaluronic acid is preferably less than or equal to 6% by mass volume, preferably 1-4% by mass, and more preferably 2% by mass. In the present invention, the amount of the physiological saline may be used according to the standard defined in the art.

In the present invention, the term "pharmaceutically acceptable" generally means that the excipients and the like are generally nontoxic, safe, and suitable for use by patients. The "patient" is preferably a mammal, more preferably a human.

Preferably, the ophthalmic preparation can be in the form of products which are conventionally applied to eyes in the field, such as drops, for example, eye drops, sprays, gels, ophthalmic liposomes, and the like.

Preferably, the ophthalmic formulation is administered 1-3 times daily, preferably at a rate of 0.3-0.8nmol protein per eye.

Preferably, the ophthalmic formulation is administered 2 times daily, 1 drop at a time, for 3 months.

Preferably, the ophthalmic formulation is administered 1 time daily, 1 drop at a time, for 5 days.

Preferably, the ophthalmic formulation is administered 2 times daily, 1 drop at a time, for 2 weeks.

In a preferred embodiment, the ophthalmic formulation consists of 10 μmol/L transthyretin, 2% (mass volume percent) hyaluronic acid, 10 μmol/L diclofenac sodium and physiological saline.

In a preferred embodiment, the ophthalmic formulation consists of 10 μmol/L transthyretin, 2% (mass volume percent) hyaluronic acid, 5 μmol/L vitamin A, 5% (v/v) Tween 80 and physiological saline.

In a preferred embodiment, the ophthalmic formulation consists of 10. mu. mol/L transthyretin, 2% (mass volume percent) hyaluronic acid, 5. mu. mol/L luteolin and physiological saline.

Preferably, the ophthalmic preparation is an ophthalmic preparation for inhibiting ocular retinal leakage and/or reducing the number of retinal neovessels, and the ophthalmic preparation is preferably an ophthalmic preparation for treating diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity.

In order to solve the above-mentioned technical problems, the second aspect of the present invention provides a method for producing an ophthalmic preparation according to the first aspect of the present invention by mixing a compound according to the first aspect of the present invention with said transthyretin.

In order to solve the above technical problems, the third aspect of the present invention provides the use of the ophthalmic preparation according to the first aspect of the present invention for the preparation of a medicament for inhibiting ocular retinal leakage and/or reducing retinal neovascularization count; preferably in the preparation of a medicament for the treatment of diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity.

As used herein, the term "solvate" refers to a substance formed by combining a compound of the present invention with a stoichiometric or non-stoichiometric amount of a solvent. The solvent molecules in the solvate may be present in an ordered or unordered arrangement. Such solvents include, but are not limited to: water, methanol, ethanol, and the like.

In the present invention, "pharmaceutically acceptable salt" and "solvate" in "solvate of pharmaceutically acceptable salt" are as defined above, and refer to a substance formed by combining compound 1 of the present invention, 2 prepared from a relatively nontoxic, pharmaceutically acceptable acid or base, and a stoichiometric or non-stoichiometric amount of a solvent. The "solvate of pharmaceutically acceptable salt" includes but is not limited to hydrochloride monohydrate of the adjuvant of the present invention.

In the present invention, the "compound", "pharmaceutically acceptable salt", "glycoside", "solvate" and "solvate of pharmaceutically acceptable salt" may exist in a crystalline form or an amorphous form. The term "crystal form" refers to a form in which ions or molecules are arranged strictly periodically in a three-dimensional space in a defined manner and have a periodic recurring pattern at a distance; due to the above described periodic arrangement, various crystal forms, i.e. polymorphism, may exist. The term "amorphous" refers to a state in which ions or molecules are distributed in a disordered manner, i.e., the ions and molecules do not have a periodic arrangement.

In the present invention, the term "comprising" may mean that other components exist in addition to the components listed below; in some cases, it may also be said to "consist of … …," i.e., including only the ingredients listed below and no other ingredients present.

In the invention, the transthyretin is a tetramer carrier protein and can transport thyroid hormone in plasma and cerebrospinal fluid. Transthyretin entry into cells was found to be mediated by the high density lipoprotein receptor SRB1 (Landers, K.A., et al, Transthyretin uptake in planar cells is regulated by the high-sensitivity lipid receptor, weaver receptor class B member 1.Mol Cell endothelial Cell 2018.474: p.89-96). From a structural point of view (see figure 1 for three-dimensional structure), the tetrameric surface of transthyretin has significant hydrophilic domains (dark color) and hydrophobic domains (light color), and the core hydrophobic domain of transthyretin is capable of carrying strongly hydrophobic thyroxine molecules across various cells (see figure 2). In addition, the amino acid sequences of transthyretin from different species are highly conserved, with the amino acid sequence similarity of human transthyretin to SD rat and C57BL/6 mouse-derived transthyretin being > 95% (see FIG. 3).

In the present invention, the mentioned compounds and the like can be generally obtained commercially, for example, from the national drug group, the welfare technologies ltd, and the like.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the transthyretin in the ophthalmic preparation of the present invention can be synergistically combined with the specific compound, so that the therapeutic effect on ocular diseases associated with ocular angiogenesis and/or ocular retinal leakage can be significantly improved when the ophthalmic preparation is used for treating ocular diseases. In a preferred embodiment of the present invention, the ophthalmic preparation of the present invention not only can effectively treat DR, AMD and ROP, but also can nourish the eyes, and has a certain relieving effect on retinal leakage and neovascularization increase of the eyes due to other reasons.

Drawings

FIG. 1 shows the three-dimensional structure (PDB ID: 1ICT) of transthyretin (TTR).

Figure 2 shows that the TTR core hydrophobic domain carries a strongly hydrophobic thyroxine molecule across various cells.

FIG. 3 shows that the similarity of human TTR and SD rat, C57BL/6 mouse and rabbit derived TTR amino acid sequence is > 95%, wherein SD rat derived TTR amino acid sequence is shown as SEQ ID NO. 5, and C57BL/6 mouse derived TTR amino acid sequence is shown as SEQ ID NO. 6.

Figure 4A shows that the protein structure is TTR dimer, the diclofenac ligand molecule is indicated using an arrow, and one molecule of TTR dimer can bind two molecules of diclofenac.

Figure 4B shows the interaction of diclofenac with TTR amino acid residues.

FIG. 5A shows that vitamin A1 was found to bind stably to the hydrophobic channels of TTR multimers by molecular modeling using Discovery studio software, where the protein structure is TTR dimer, and the vitamin A1 ligand molecule is indicated by the arrow, and one molecule of TTR dimer can bind to one molecule of vitamin A1.

Fig. 5B shows the interaction of vitamin a1 with TTR amino acid residues.

FIG. 6A shows that vitamin A2 can stably bind to the hydrophobic channels of a TTR multimer in molecular modeling using Discovery studio software, where the protein structure is TTR dimer, and one molecule of TTR dimer can bind to one molecule of vitamin A2, as indicated by the arrows in the vitamin A2 ligand molecule.

Fig. 6B shows the interaction of vitamin a2 with TTR amino acid residues.

FIG. 7A shows that luteolin can be stably bound to a TTR multimer as determined by molecular simulation using Discovery studio software, wherein the protein structure is TTR dimer, and the luteolin ligand molecule is indicated by an arrow, and one molecule of TTR dimer can bind one molecule of luteolin.

Figure 7B shows the interaction of luteolin with TTR amino acid residues.

FIG. 8A shows that sulfamethoxazole can be stably bound to TTR multimers by molecular simulation using Discovery studio software, where the protein structure is TTR dimer, the sulfamethoxazole ligand molecule is indicated by an arrow, and one molecule of TTR dimer can bind to one molecule of sulfamethoxazole.

FIG. 8B shows the interaction of sulfamethoxazole with TTR amino acid residues.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.

Example 1 recombinant preparation of human transthyretin (TTR)

(1) Construction of the recombinant plasmid pETx-rhaPBAD-ttr: the pET-21a plasmid (purchased from ATCC China center for culture Collection) is reconstructed (the difference of the plasmid obtained after reconstruction and the original pET-21a plasmid in sequence is about 75%, the specific sequence is shown as SEQ ID NO: 4), rhaPBAD promoter (Rhamngtang inducible) is used for replacing T7 promoter, meanwhile, the rhaPBAD promoter is connected with human TTR optimized nucleic acid sequence (shown as SEQ ID NO:2, the amino acid sequence of TTR is shown as SEQ ID NO: 1), and the whole nucleic acid sequence of the obtained plasmid is shown as SEQ ID NO: 3. The construction was successful after sequencing verification (the sequencing company is Nanjing King Shirui Biotechnology Co., Ltd.).

(2) Expression and purification of recombinant human TTR: transforming the pETx-rhaPBAD-ttr plasmid constructed in the step (1) into E.coli BL21(DE3) cells, culturing the obtained recombinant E.coli BL21(DE3) in LB culture medium to prepare seed liquid, inoculating the seed liquid into 5L TB culture medium at the inoculation amount of 5 percent and the temperature of 37 ℃, the rotation speed of a stirring paddle of 150rpm, and culturingTo OD₆₀₀1.5-2.0; adding 0.4-2% (mass volume percent) rhamnose to induce for 16-20h (table 1). Crushing thallus with high pressure homogenizer, and passing the supernatant through nickel (Ni)⁺) And (4) performing column chromatography to obtain the humanized TTR. Endotoxin adsorption column (Pierce) was used^TMHigh Capacity endo toxin Removal Spin Columns, ThermoFisher) and the residual bacteria were removed through a 0.22 μm pore size filter. The obtained production amounts of TTR protein of human origin are shown in Table 1. At OD₆₀₀When the concentration is 1.5-2.0, 0.4-2% rhamnose is used for inducing for 16-20h, and the obtained protein yield is more than or equal to 50mg/g wet thallus.

TABLE 1 recombinant expression of TTR of human origin

Example 2 computer modeling of TTR binding morphology to individual ligand molecules

Referring to the PDB database, a model of static co-crystallization of TTR dimer with diclofenac acid (PDF: 3CFQ) is shown in FIG. 4A. In fig. 4A, the protein structure is TTR dimer, molecules of diclofenac ligand are indicated using arrows, and one molecule of TTR dimer can bind two molecules of diclofenac. The interaction of diclofenac with TTR amino acid residues is shown in figure 4B.

Molecular modeling with Discovery studio software found that vitamin a1 (retinol) was able to stably bind to the hydrophobic channels of TTR multimers. The protein structure shown in fig. 5A is TTR dimer, and the vitamin a1 ligand molecule is indicated using arrows, and one molecule of TTR dimer can bind one molecule of vitamin a 1. The interaction of vitamin a1 with TTR amino acid residues is shown in fig. 5B.

Molecular modeling with Discovery studio software found that vitamin a2 (3-dehydroretinol) was able to stably bind to the hydrophobic channels of TTR multimers. The protein structure shown in fig. 6A is TTR dimer, and the vitamin a2 ligand molecule is indicated using arrows, and one molecule of TTR dimer can bind one molecule of vitamin a 2. The interaction of vitamin a2 with TTR amino acid residues is shown in fig. 6B.

Molecular simulation by Discovery studio software found that luteolin was able to stably bind to TTR multimers. The protein structure shown in fig. 7A is TTR dimer, and the luteolin ligand molecule is indicated using 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 7B.

From the above results, it can be seen that the interaction force between TTR and these ligand molecules is mainly non-covalent interaction force such as van der waals force, hydrogen bonding, hydrophobic interaction, etc., and it is not necessary to change the chemical structure between TTR and the ligand in nature.

Example 3 determination of dynamic specific binding parameters of TTR to various potential ligand molecules

Determination of the affinity binding equilibrium dissociation constant K of TTR to the respective ligand molecule mentioned in example 2 or a salt thereof using nano ITC (TA)_d100. mu. mol/L of each of the above ligand solutions was dropped into 10. mu. mol/L of TTR solution (1000. mu.L) at a rate of 1. mu.L/min, and the affinity binding equilibrium dissociation constant K was calculated using a software built in the solution_d(Table 2).

TABLE 2 affinity binding equilibrium dissociation constants for various ligand molecules to TTR

As shown in Table 2, the affinity binding equilibrium dissociation constant of various ligand molecules and TTR is close to 10^-8mol/L, which is close to the ability of monoclonal antibody to recognize single epitope, indicates that the ligand molecule can specifically recognize and bind with TTR.

Example 4 human TTR-ligand molecule complexes Cross corneal Barrier into the vitreous and fundus

The human TTR prepared in example 1 was prepared as 10. mu. mol/L (containing physiological saline, 2% low molecular weight hyaluronic acid and 10. mu. mol/L diclofenac sodium/5. mu. mol/L vitamin A + 5% (v/v) Tween 80/5. mu. mol/L luteolin (the addition ratio was the result of computer simulation in example 2), C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) were respectively dropped into eye, sacrificed after 3-72 hours, the vitreous body and fundus sample were taken to extract protein, rabbit anti-His-tag antibody was used as the primary antibody, donkey anti-rabbit antibody was used as the secondary antibody, the content of human TTR in the vitreous body of C57BL/6 mice and SD rats and fundus sample was measured by ELISA. the second part, page 115, item of diclofenac sodium, of China pharmacopoeia 2020 edition, measuring the content of diclofenac sodium in the sample; determining the content of the vitamin A in the sample according to the entry of the vitamin A on page 1472 of the second part of the Chinese pharmacopoeia 2020 edition; the content of Luteolin in the sample is determined by reference to British pharmacopoeia BP2017, entry Luteolin-7-glucoside.

After eye dropping, the half-life of the human TTR and each ligand molecule in the vitreous body and fundus sample of C57BL/6 mouse and SD rat is close to 60h, which shows that the human TTR and each ligand molecule can effectively exist for 60h in the vitreous body and fundus sample, and the sufficient treatment concentration and treatment time are provided (Table 3).

TABLE 3 introduction of human TTR and ligand molecules across the corneal barrier of C57BL/6 mice and SD rats into the vitreous and fundus oculi

Example 5 eye drop treatment of DR (diabetic retinopathy) SD rats with human TTR-ligand molecule Complex

SD rats of 8 weeks old are 200-250g in weight, fasted for 12-18h, injected with 2% STZ (60mg/kg) in the abdominal cavity, collected by tail cutting after 48h and 72h, tested by blood glucose test paper is higher than 16.7mM, and successfully molded to obtain DR SD rats. DR SD rats are divided into 6 groups, 1 group is DR SD rats without any treatment (5 rats), the other 5 groups are 5 rats per group, the left eye and the right eye are respectively dripped 2 times a day, and 30 mu L of each time, wherein the left eye is used for dripping the human TTR prepared in example 1, or the human TTR + ligand eye prepared in example 1 is used for dripping, specifically 10 mu mol/L of the human TTR (physiological saline solution + 2% hyaluronic acid), 10 mu mol/L of the human TTR (physiological saline solution + 2% hyaluronic acid +10 mu mol/L diclofenac sodium), 10 mu mol/L of the human TTR (physiological saline solution + 2% hyaluronic acid +5 mu mol/L vitamin A + 5% Tween 80), 10 mu mol/L of the human TTR (physiological saline solution + 2% hyaluronic acid +5 mu mol/L luteolin) are respectively dripped, the right eye is prepared by adding normal saline solution and 2% hyaluronic acid, or adding normal saline solution and 2% hyaluronic acid and ligand, and as a control, the right eye is prepared by adding normal saline solution and 2% hyaluronic acid, normal saline solution and 2% hyaluronic acid and 10 μmol/L diclofenac sodium, normal saline solution and 2% hyaluronic acid and 5 μmol/L vitamin A and 5% Tween 80, and normal saline solution and 2% hyaluronic acid and 5 μmol/L luteolin. In addition, another 1 group of normal SD rats served as controls (5). After all SD rats are continuously raised for 3 months, retinas are stripped respectively, Evans Blue staining is carried out to observe the retinal vessel leakage condition, and Trypsin enzymolysis is carried out to observe the neovascular density.

The results show that after the SD rats are bred for 3 months by STZ induction, compared with normal controls, the retinal vascular leakage and the number of new blood vessels of a control group of the DR SD rats without any treatment are obviously improved, the eyeball retinal leakage phenomenon of a group of which human-derived TTR or human-derived TTR/diclofenac sodium, human-derived TTR/vitamin A and human-derived TTR/luteolin is dropwise added is obviously inhibited, the number of new blood vessels of the retina is obviously reduced, and the clinical pathological phenomenon of DR is relieved (Table 4).

TABLE 4 treatment of STZ induced SD rat DR pathological conditions with human TTR/human TTR-ligand molecular complexes

EXAMPLE 6 eye drop treatment of AMD (age-related macular degeneration) C57BL/6 mice with TTR-ligand molecule complexes of human origin

9 weeks old C57/BL6 mice were photocoagulated to the retina with krypton laser (647nm) at 360mW, 50 μm in diameter for 0.05s at 8 photocoagulation points per eye to induce choroidal neovascularization and progressive proliferation to the retina to obtain AMD C57BL/6 mice. AMD C57BL/6 mice were divided into 6 groups, 1 group was AMD C57BL/6 mice without any treatment (5 mice), and the other 5 groups were 5 mice per group, and left and right eyes were each added dropwise 2 times a day, 30. mu.L each time, wherein left eyes were eye drops of the human TTR prepared in example 1, or eye drops of the human TTR + ligand prepared in example 1, specifically 10. mu. mol/L human TTR (physiological saline solution + 2% hyaluronic acid), 10. mu. mol/L human TTR (physiological saline solution + 2% hyaluronic acid + 10. mu. mol/L diclofenac sodium), 10. mu. mol/L human TTR (physiological saline solution + 2% hyaluronic acid + 5. mu. mol/L vitamin A + 5% Tween 80), 10. mu. mol/L human TTR (physiological saline solution + 2% hyaluronic acid + 5. mu. mol/L luteolin) respectively, the right eye is prepared by dropping normal saline solution + 2% hyaluronic acid, or normal saline solution + 2% hyaluronic acid + ligand, and as a control, the right eye is specifically prepared by dropping normal saline solution + 2% hyaluronic acid, normal saline solution + 2% hyaluronic acid +10 μmol/L diclofenac sodium, normal saline solution + 2% hyaluronic acid +5 μmol/L vitamin A + 5% Tween 80, and normal saline solution + 2% hyaluronic acid +5 μmol/L luteolin. In addition, 1 additional group of normal C57BL/6 mice served as controls (5). After 2 weeks of eye drop, the animals were sacrificed, the retinas were stripped and subjected to Evans Blue staining for retinal vessel leakage and Trypsin enzymatic digestion for neovascular density.

The results are shown in Table 5, which shows that compared with the normal control group, the retinal vascular leakage and the number of new blood vessels of the control group without any treatment of AMD C57BL/6 mice are obviously improved; and the retinal leakage condition and the neovascularization condition of the human TTR or the human TTR/diclofenac sodium, the human TTR/vitamin A and the human TTR/luteolin group are obviously relieved.

TABLE 5 treatment of AMD pathological conditions in mice with laser retinal photocoagulation induced C57BL/6 by TTR/ligand molecule complexes of human origin

Example 7 eye drop treatment of ROP (retinopathy of prematurity) SD rats with human TTR-ligand molecule Complex

One week after birth (SD rat) is placed in a hyperbaric oxygen chamber for feeding, and a normal control group is placed in a normal environment for feeding (normal control group, 5 animals). The rat was raised in a hyperbaric oxygen chamber for 5 days and then taken out to obtain ROP SD rat. All ROP SD rats were kept in normal environment for 5 days together with normal controls. In the 5 days, the ROP SD rats were divided into 6 groups, 1 group was ROP SD mice without any treatment (5 mice), and the other 5 groups were 5 mice/group, and the left eye and the right eye were each dropped 1 times a day by 30. mu.L each, wherein the left eye was dropped with the human TTR prepared in example 1 or with the human TTR + ligand prepared in example 1, specifically with 10. mu. mol/L human TTR (physiological saline solution + 2% hyaluronic acid), 10. mu. mol/L human TTR (physiological saline solution + 2% hyaluronic acid + 10. mu. mol/L diclofenac sodium), 10. mu. mol/L human TTR (physiological saline solution + 2% hyaluronic acid + 5. mu. mol/L vitamin A + 5% Tween 80), 10. mu. mol/L human TTR (physiological saline solution + 2% hyaluronic acid + 5. mu. mol/L gals luteolin) respectively, the right eye is prepared by adding normal saline solution and 2% hyaluronic acid, or adding normal saline solution and 2% hyaluronic acid and ligand, and as a control, the right eye is prepared by adding normal saline solution and 2% hyaluronic acid, normal saline solution and 2% hyaluronic acid and 10 μmol/L diclofenac sodium, normal saline solution and 2% hyaluronic acid and 5 μmol/L vitamin A and 5% Tween 80, and normal saline solution and 2% hyaluronic acid and 5 μmol/L luteolin. After 5 days of normal environment feeding, the suckling mice were sacrificed and retinas were stripped for Evans Blue staining to observe retinal vascular leakage.

The results are shown in table 6, which shows that the retinal vascular leakage of the control group of ROP SD rats without any treatment is significantly improved compared to the normal control group; and the retinal leakage condition of the human TTR or human TTR/diclofenac sodium, human TTR/vitamin A and human TTR/luteolin group is remarkably relieved.

TABLE 6 pathological condition of rat suckling rat ROP induced by hyperbaric oxygen chamber treatment of TTR/ligand molecule composite derived from human

It can be seen from the data of examples 5 to 7 that even normal SD rats have retinal leaks worse than those of young SD rats, and that other reasons, such as experimental procedures, individual differences, etc., may cause retinal leaks and an increase in the number of new blood vessels, indicating that even normal control eyes without modeling have retinal leaks and new blood vessels. After the human TTR/diclofenac sodium or the human TTR/vitamin A is used, the traditional Chinese medicine composition can treat DR, AMD and ROP, can play a nourishing role, and has a certain relieving effect on retinal leakage and neovascularization increase of eyes caused by other reasons.

Comparative example 1:

the present embodiment is different from example 4 in that the same procedure as in example 4 was carried out for eye drop test of C57BL/6 mouse (8 weeks old) and SD rat (8 weeks old) using each ligand molecule not binding to TTR, and the results show that each ligand molecule alone cannot enter the vitreous body and fundus oculi of C57BL/6 mouse and SD rat (table 7).

TABLE 7 respective ligand molecules cross the corneal barrier of C57BL/6 mice and SD rats into the vitreous and fundus

Comparative example 2:

in the same manner as in example 5, data of the results after the right eye was dropped are shown in Table 8. The results show that the single eye drop of each ligand molecule failed to improve the pathological conditions of STZ-induced SD rat DR (table 8).

TABLE 8 treatment of STZ induced DR pathology in SD rats with eye drops of each ligand alone

Comparative example 3:

in the same manner as in example 6, data of the results after the right eye was dropped are shown in Table 9. The results show that the single eye drop of each ligand molecule failed to improve the pathological condition of AMD in the laser retinal photocoagulation induced C57BL/6 mice (Table 9).

TABLE 9 eye drop treatment of AMD pathological conditions induced by laser retinal photocoagulation of C57BL/6 mice with ligands alone

Comparative example 4:

in the same manner as in example 7, data of the results after the right eye was dropped are shown in Table 10. The results show that the pathological condition of hyperbaric oxygen chamber-induced rat suckers cannot be improved by the single eye drop of each ligand molecule (table 10).

TABLE 10 treatment of pathological conditions of ROP induced in SD rat by hyperbaric oxygen chamber with eye drops of each ligand alone

Comparative example 5:

sulfamethoxazole (sulfamethoxazole) has wide antibacterial spectrum and strong antibacterial effect, can block the growth of bacteria, and is particularly effective to staphylococcus and escherichia coli; is suitable for respiratory system, urinary system and intestinal tract infection; it is mainly used for treating fowl cholera. It can be used for preparing eye drop such as compound sulfamethoxazole sodium eye drop (the product is compound preparation, every 10 ml contains sulfamethoxazole sodium 400 mg, aminocaproic acid 200 mg, dipotassium glycyrrhizinate 10 mg, chlorphenamine maleate 2 mg, and is mainly used for bacterial conjunctivitis, hordeolum and bacterial blepharitis caused by sensitive bacteria).

Molecular modeling by Discovery studio software found that sulfamethoxazole was able to stably bind to TTR multimers. The protein structure shown in FIG. 8A is TTR dimer, the sulfamethoxazole ligand molecule is indicated with an arrow, and one molecule of TTR dimer can bind one molecule of sulfamethoxazole. The interaction of sulfamethoxazole with TTR amino acid residues is shown in FIG. 8B.

Referring to the procedure of example 3, 100. mu. mol/L sulfamethoxazole sodium salt solution was added dropwise at a rate of 1. mu.L/min to 10. mu. mol/L TTR solution (1000. mu.L), and the affinity binding equilibrium dissociation constant Kd was calculated to be 7.03X 10 using a software built-in^-8mol/L。

When rats and mice were eyedropped with TTR/sulfamethoxazole sodium salt for 3 to 72 hours according to the procedure of example 4, corneal injury occurred in rats and mice, that is, corneal burn occurred when sulfamethoxazole sodium salt was used in combination with TTR, and there was no biological safety. Therefore, after ligands which have the same biological activity and the anti-inflammatory effect and can be combined with TTR are screened out by using the Discovery studio software molecular simulation, the therapeutic effect can not be improved in all cases when the experiment verification is carried out.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Tongyan (Shanghai) medical Instrument Co., Ltd

<120> an ophthalmic preparation, a preparing method and applications thereof

<130> P20014219C

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 127

<212> PRT

<213> Homo sapiens

<400> 1

Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val

1 5 10 15

Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val Ala Val His Val

20 25 30

Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys

35 40 45

Thr Ser Glu Ser Gly Glu Leu His Gly Leu Thr Thr Glu Glu Glu Phe

50 55 60

Val Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys

65 70 75 80

Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu Val Val Phe Thr

85 90 95

Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser

100 105 110

Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn Pro Lys Glu

115 120 125

<210> 2

<211> 381

<212> DNA

<213> Artificial Sequence

<220>

<223> TTR mature fragment optimized nucleic acid sequence

<400> 2

ggtccgaccg gtaccggtga atctaaatgc ccgctgatgg ttaaagttct ggacgctgtt 60

cgtggttctc cggctatcaa cgttgctgtt cacgttttcc gtaaagctgc tgacgacacc 120

tgggaaccgt tcgcttctgg taaaacctct gaatctggtg aactgcacgg tctgaccacc 180

gaagaagaat tcgttgaagg tatctacaaa gttgaaatcg acaccaaatc ttactggaaa 240

gctctgggta tctctccgtt ccacgaacac gctgaagttg ttttcaccgc taacgactct 300

ggtccgcgtc gttacaccat cgctgctctg ctgtctccgt actcttactc taccaccgct 360

gttgttacca acccgaaaga a 381

<210> 3

<211> 4693

<212> DNA

<213> Artificial Sequence

<220>

<223> plasmid Overall nucleic acid sequence

<400> 3

aattcttaag aaggagatat acatatgaga ggatcgcatc atcatcatca tcatggatcc 60

ggtccgaccg gtaccggtga atctaaatgc ccgctgatgg ttaaagttct ggacgctgtt 120

cgtggttctc cggctatcaa cgttgctgtt cacgttttcc gtaaagctgc tgacgacacc 180

tgggaaccgt tcgcttctgg taaaacctct gaatctggtg aactgcacgg tctgaccacc 240

gaagaagaat tcgttgaagg tatctacaaa gttgaaatcg acaccaaatc ttactggaaa 300

gctctgggta tctctccgtt ccacgaacac gctgaagttg ttttcaccgc taacgactct 360

ggtccgcgtc gttacaccat cgctgctctg ctgtctccgt actcttactc taccaccgct 420

gttgttacca acccgaaaga atgaaagctt ctgttttggc ggatgagaga agattttcag 480

cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg 540

cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc 600

cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac 660

gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc 720

tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag 780

ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag aaggccatcc 840

tgacggatgg cctttttgcg tttctacaaa ctcttttgtt tatttttcta aatacattca 900

aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 960

aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 1020

cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 1080

ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 1140

cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 1200

ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 1260

gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 1320

gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 1380

acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 1440

cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 1500

acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 1560

ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 1620

ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 1680

gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 1740

atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 1800

ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 1860

attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 1920

ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 1980

aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 2040

aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 2100

ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 2160

tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 2220

ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 2280

cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 2340

agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 2400

gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 2460

ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 2520

tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 2580

tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 2640

cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 2700

tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 2760

gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 2820

atatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac 2880

tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga 2940

cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 3000

cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg 3060

gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc 3120

cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt 3180

aagggcggtt ttttcctgtt tggtcacttg atgcctccgt gtaaggggga atttctgttc 3240

atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat 3300

gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg 3360

gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt 3420

ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct 3480

gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct 3540

caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca 3600

ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg 3660

atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg 3720

gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc 3780

cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg 3840

cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga 3900

caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg 3960

cataaatcgc cgtgacgatc agcggtccag tgatcgaagt taggctggta agagccgcga 4020

gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca 4080

acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc 4140

ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa 4200

tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg 4260

cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc 4320

ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa 4380

agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga 4440

ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc cttatgcgac tcctgcatta 4500

ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat 4560

gcatcgatca ccacaattca gcaaattgtg aacatcatca cgttcatctt tccctggttg 4620

ccaatggccc attttcctgt cagtaacgag aaggtcgcga attcaggcgc tttttagact 4680

ggtcgtaatg aac 4693

<210> 4

<211> 4311

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of pET-21a plasmid after reconstruction

<400> 4

aattcttaag aaggagatat acatatggga tcccatcatc atcatcatca ttgactgcag 60

ccaagcttct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa 120

cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct 180

gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc 240

catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg 300

ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc 360

gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc 420

ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt 480

tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 540

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 600

tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 660

aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 720

aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 780

tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc 840

aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 900

tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 960

ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 1020

taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 1080

agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 1140

caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 1200

tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 1260

gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 1320

cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 1380

caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 1440

ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 1500

aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 1560

gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1620

atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1680

tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 1740

gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 1800

actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 1860

gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 1920

agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 1980

ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 2040

aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 2100

cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2160

gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2220

cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 2280

cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 2340

gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt 2400

attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac 2460

aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg 2520

gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 2580

ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 2640

ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg 2700

tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga 2760

agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg 2820

gtcacttgat gcctccgtgt aagggggaat ttctgttcat gggggtaatg ataccgatga 2880

aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac 2940

gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg 3000

gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc 3060

ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt 3120

acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc 3180

agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac 3240

cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg 3300

acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata 3360

tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa 3420

ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc 3480

ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac 3540

aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag 3600

cggtccagtg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg 3660

atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc 3720

ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa 3780

gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg 3840

tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc 3900

aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca 3960

gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc 4020

gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg 4080

catcggtcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt 4140

gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc atcgatcacc acaattcagc 4200

aaattgtgaa catcatcacg ttcatctttc cctggttgcc aatggcccat tttcctgtca 4260

gtaacgagaa ggtcgcgaat tcaggcgctt tttagactgg tcgtaatgaa c 4311

<210> 5

<211> 127

<212> PRT

<213> Rattus norvegicus

<400> 5

Gly Pro Gly Gly Ala Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val

1 5 10 15

Leu Asp Ala Val Arg Gly Ser Pro Ala Val Asp Val Ala Val Lys Val

20 25 30

Phe Lys Lys Thr Ala Asp Gly Ser Trp Glu Pro Phe Ala Ser Gly Lys

35 40 45

Thr Ala Glu Ser Gly Glu Leu His Gly Leu Thr Thr Asp Glu Lys Phe

50 55 60

Thr Glu Gly Val Tyr Arg Val Glu Leu Asp Thr Lys Ser Tyr Trp Lys

65 70 75 80

Ala Leu Gly Ile Ser Pro Phe His Glu Tyr Ala Glu Val Val Phe Thr

85 90 95

Ala Asn Asp Ser Gly His Arg His Tyr Thr Ile Ala Ala Leu Leu Ser

100 105 110

Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Ser Asn Pro Gln Asn

115 120 125

<210> 6

<211> 127

<212> PRT

<213> Mus musculus

<400> 6

Gly Pro Ala Gly Ala Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val

1 5 10 15

Leu Asp Ala Val Arg Gly Ser Pro Ala Val Asp Val Ala Val Lys Val

20 25 30

Phe Lys Lys Thr Ser Glu Gly Ser Trp Glu Pro Phe Ala Ser Gly Lys

35 40 45

Thr Ala Glu Ser Gly Glu Leu His Gly Leu Thr Thr Asp Glu Lys Phe

50 55 60

Val Glu Gly Val Tyr Arg Val Glu Leu Asp Thr Lys Ser Tyr Trp Lys

65 70 75 80

Thr Leu Gly Ile Ser Pro Phe His Glu Phe Ala Asp Val Val Phe Thr

85 90 95

Ala Asn Asp Ser Gly His Arg His Tyr Thr Ile Ala Ala Leu Leu Ser

100 105 110

Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Ser Asn Pro Gln Asn

115 120 125

<210> 7

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> human TTR-CL

<400> 7

Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val

1 5 10 15

Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val Ala Val His Val

20 25 30

Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys

35 40 45

Thr Ser Glu Ser Gly Glu Leu His Gly Leu Thr Thr Glu Glu Glu Phe

50 55 60

Val Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys

65 70 75 80

Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu Val Val Phe Thr

85 90 95

Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser

100 105 110

Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val

115 120

Claims (13)

1. An ophthalmic formulation for inhibiting ocular retinal leakage and/or reducing retinal neovascularity, comprising diclofenac or a pharmaceutically acceptable salt thereof, and transthyretin; the transthyretin is protein consisting of amino acids shown in SEQ ID NO. 1; the content of the transthyretin is 5-20 mu mol/L; the content of the diclofenac or the pharmaceutically acceptable salt thereof is 5-20 mu mol/L.

2. The ophthalmic formulation of claim 1, wherein said pharmaceutically acceptable salt is diclofenac sodium.

3. The ophthalmic formulation of claim 1, wherein said transthyretin is present in an amount of 10-20 μmol/L.

4. The ophthalmic formulation of claim 3, wherein said transthyretin is present in an amount of 10, 15 or 20 μmol/L and said diclofenac, or a pharmaceutically acceptable salt thereof, is present in an amount of 10 μmol/L.

5. The ophthalmic formulation of claim 1, wherein the nucleotide sequence encoding said transthyretin is set forth in SEQ ID No. 2.

6. The ophthalmic formulation of claim 1, further comprising a pharmaceutically acceptable excipient;

and/or, the ophthalmic preparation is drops, spray, gel or ophthalmic liposome;

and/or, the ophthalmic formulation is administered from 1 to 3 times daily.

7. The ophthalmic formulation of claim 6, wherein said ophthalmic formulation is administered 2 times daily, 1 drop at a time, for 3 months; or, the ophthalmic formulation is administered 1 time daily, 1 drop at a time, for 5 days; or, the ophthalmic formulation is administered 2 times daily, 1 drop at a time, for 2 weeks;

and/or the auxiliary material is normal saline, a surfactant and/or hyaluronic acid.

8. The ophthalmic formulation of claim 7, wherein the surfactant is tween 80;

and/or, the content of the surfactant is 5%;

and/or the content of the hyaluronic acid is less than or equal to 6 percent;

and/or the drops are eye drops;

and/or, the ophthalmic formulation is administered at a rate of 0.3 to 0.8nmol protein per eye per administration.

9. The ophthalmic formulation of claim 8, wherein the hyaluronic acid is present in an amount of 1-4%.

10. The ophthalmic formulation of claim 9, wherein the hyaluronic acid is present in an amount of 2%.

11. A process for the preparation of an ophthalmic formulation as claimed in any one of claims 1 to 10, characterized in that diclofenac or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 10 is mixed with said transthyretin.

12. Use of an ophthalmic formulation according to any one of claims 1 to 10 for the preparation of a medicament for inhibiting retinal leakage and/or reducing the number of retinal neovasculature in the eye.

13. The use according to claim 12, for the preparation of a medicament for the treatment of diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity.

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