APPLICATION OF TRANSTHYRETIN IN ENTERING EYE AND PREPARING DROP

Abstract

The present invention provides an application of transthyretin serving as a carrier for a protein and/or polypeptide drug to enter an eye through an eye barrier. The transthyretin is a protein consisting of amino acid as shown in SEQ ID NO: 1 or a mutation thereof or a modification thereof. Further provided are an application of transthyretin and/or a fusion protein of the transthyretin and a drug in preparation of a drops, and a drops. The drug is a protein and/or polypeptide drug. The transthyretin has good biocompatibility and safety in human bodies, can effectively convey a foreign protein and/or polypeptide into the eye, and achieves an effect of treating eye diseases.

Claims

1. A method for transferring a protein or polypeptide medicament into an eye through an ocular barrier using transthyretin as a carrier, wherein the transthyretin is represented by (a), (b) or (c): (a) a protein consisting of an amino acid sequence of SEQ ID NO: 1; (b) a protein derived from (a) with an inhibitory function of neovascularization, which is shown by a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (a); (c) a protein having a sequence with a hydrophilic modification or hydrophobic modification in the amino acid sequence of (a) or (b).

2. The method of claim 1, wherein in (b), the protein derived from (a) is a protein in which 22 amino acids or 25 amino acids are substituted in, or 5 amino acids are deleted from the amino acid sequence of (a); or, in (c), the hydrophilic modification or hydrophobic modification occurs at the cysteine at position 10 of the amino acid sequence of (a).

3. The method of claim 1, wherein the transthyretin is expressed with the protein or polypeptide medicament as a fusion; and the fusion is preferably a fusion that the protein or polypeptide medicament are fused at the N-terminus or C-terminus of the transthyretin; preferably, the transthyretin is expressed with the protein or polypeptide medicament as a fusion in a microbial cell, followed by a purification of the fusion; wherein the purification is preferably to remove endotoxin by an endotoxin absorption column and then remove residue bacteria by a filter membrane with a pore size of 0.22 μm; or, the protein or polypeptide medicament comprises lysozyme, albumin or EGFR antibody, with a molecular weight of no more than 45 kDa; the lysozyme is preferably hen egg white lysozyme with GenBank Accession No.: AAL69327.1; the albumin is preferably ovalbumin; or, the protein or polypeptide medicament comprises a protein or polypeptide medicament for treating ocular diseases associated with ocular retina leakage or retinal neovascularization such as diabetic retinopathy, age-related macular degeneration or retinopathy of prematurity.

4. The method of claim 1, wherein the transthyretin is encoded by a nucleotide sequence of SEQ ID NO: 2; or, the transthyretin is expressed by a recombinant expression vector, wherein the recombinant expression vector has a plasmid backbone comprising a rhamnose inducible promoter, preferably a rhaPBAD promoter; or, the transthyretin is expressed by a recombinant expression vector, and the recombinant expression vector has a plasmid backbone of pET-21a or a vector having 25% or more homology therewith, and the vector having 25% or more homology therewith preferably has a sequence of SEQ ID NO: 8; or, the recombinant plasmid expressing transthyretin has a nucleotide sequence of SEQ ID NO: 3; or, the transthyretin is expressed in a microbial cell, and preferably followed by a purification of the transthyretin; the microbial cell is preferably an E. coli, and the E coli preferably comprises E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5α, E. coli K12 or E. coli TOP10; the purification is preferably to remove endotoxin by an endotoxin absorption column and then remove residue bacteria by a filter membrane with a pore size of 0.22 μm; or, the transthyretin is expressed by culturing the transformant comprising a gene of the transthyretin until the bacteria obtained reaches an OD.sub.600 of 1.5-2.0, such as 1.6, 1.7, 1.8 or 1.9; or, expression of the transthyretin is induced by a reagent for inducible expression, wherein the reagent for inducible expression has a mass volume percentage of 0.1-2%, such as 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.8%, 1.2% or 1.6%, the expression is preferably induced for a period of 8-20 h, such as 10 h, 12 h, 14 h, 16 h, 17 h, 18 h or 19 h; the reagent for inducible expression is preferably rhamnose or IPTG.

5. A method for preparing a drops comprising transthyretin or a fusion protein consisting of a transthyretin and a medicament, wherein the medicament is protein or polypeptide medicament, and the transthyretin is represented by (a), (b) or (c): (a) a protein consisting of an amino acid sequence of SEQ ID NO: 1; (b) a protein derived from (a) with an inhibitory function of neovascularization, which is shown by a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (a); (c) a protein having a sequence with a hydrophilic modification or hydrophobic modification in the amino acid sequence of (a) or (b).

6. The method of claim 5, wherein in (b), the protein derived from (a) is a protein in which 22 amino acids or 25 amino acids are substituted in, or 5 amino acids are deleted from the amino acid sequence of (a); or, in (c), the hydrophilic modification or hydrophobic modification occurs at the cysteine at position 10 of the amino acid sequence of (a).

7. The method of claim 5, wherein the fusion protein contained in the drops comprising a transthyretin and a medicament has a content of 4-30 μmol/L, preferably 10-15 μmol/L; or, the transthyretin contained in the drops has a content of 4-30 μmol/L, preferably 5-30 μmol/L, more preferably 10-20 μmol/L, such as 10, 15, 20 μmol/L; or, the drops further comprises saline; or, the drops further comprise a surfactant, wherein the surfactant, for example, is Tween 80, and preferably has a content of 5% (v/v); or, the drops further comprises a pharmaceutically acceptable excipient which is one or more selected from carboxymethyl cellulose or salts thereof, chondroitin sulfate or salts thereof, dextran, and, hyaluronic acid; wherein, the carboxymethyl cellulose or salts thereof preferably has a viscosity of 800-1200 CP; or, the chondroitin sulfate is preferably chondroitin sulfate A; or, the dextran is preferably dextran 70; or, the “salts” in the “carboxymethyl cellulose or salts thereof” or “chondroitin sulfate or salts thereof” is independently preferably sodium salts or calcium salts, such as sodium carboxymethyl cellulose or chondroitin sulfate A sodium salt; or, the hyaluronic acid has a preferred molecular weight of 10000-500000; or, the carboxymethyl cellulose or salts thereof preferably has a concentration of 0-8 mg/mL except 0, more preferably 2, 4, 6 or 8 mg/mL; or, the chondroitin sulfate or salts thereof preferably has a concentration of 0-40 mg/mL except 0, more preferably 10, 20, 30 or 40 mg/mL; or, the dextran preferably has a concentration of 0-0.8 mg/mL except 0, more preferably 0.2, 0.4, 0.6 or 0.8 mg/mL; or, the hyaluronic acid preferably has a content of no more than 6% by mass volume percentage, preferably 1-4%, more preferably 2%; or, the drops further comprises a compound, pharmaceutically acceptable salts thereof, or glycoside thereof; the compound is one or more selected from diclofenac, vitamin A and luteolin; wherein, the pharmaceutically acceptable salt is preferably a sodium salt, such as diclofenac sodium; or, the glycoside is preferably luteoloside; or, the vitamin A is preferably vitamin A1 or vitamin A2; or, the diclofenac or salts thereof has a preferred content of 5-20 μmol/L, such as 10 μmol/L; or, the vitamin A has a preferred content of 2-10 μmol/L, such as 5 μmol/L; or, the luteolin or glycoside thereof has a preferred content of 2-10 μmol/L, such as 5 μmol/L; or, the drops is preferably an eye drops; or the drops is a drops that inhibits ocular retina leakage or reduces the number of retinal neovascularization; preferably a drops that treats diabetic retinopathy, age-related macular degeneration or retinopathy of prematurity; or, the drops is administered 1-3 times per day, preferably at an amount of 0.3-0.8 nmol protein per eye at each time; or, the drops is administered twice per day, one drop each time, for 3 months; or, the drops is administered once per day, one drop each time, for 5 days; or, the drops is administered twice per day, one drop each time, for 2 weeks; or, the transthyretin is encoded by a nucleotide sequence of SEQ ID NO: 2; or, the transthyretin is expressed by a recombinant expression vector, the recombinant expression vector has a plasmid backbone comprising a rhamnose inducible promoter, preferably a rhaPBAD promoter; or, the transthyretin is expressed by a recombinant expression vector, the recombinant expression vector has a plasmid backbone of pET-21a or a vector with 25% or more homology therewith, and preferably a vector with 25% or more homology therewith has a sequence of SEQ ID NO: 8; or, the recombinant plasmid expressing transthyretin has a nucleotide sequence of SEQ ID NO: 3; or, the transthyretin is expressed in a microbial cell, and preferably followed by a purification of the transthyretin; the microbial cell is preferably an E. coli, and the E. coli preferably comprises E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5α, E. coli K12 or E. coli TOP10; the purification is preferably to remove endotoxin by an endotoxin absorption column and then remove residue bacteria by a filter membrane with a pore size of 0.22 μm; or, the transthyretin is expressed by culturing the transformant comprising a gene of the transthyretin until the bacteria obtained reaches an OD.sub.600 of 1.5-2.0, such as 1.6, 1.7, 1.8 or 1.9; or, expression of the transthyretin is induced by a reagent for inducible expression, wherein the reagent for inducible expression has a mass volume percentage of 0.1-2%, such as 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.8%, 1.2% or 1.6%, and the expression is preferably induced for a period of 8-20 h, such as 10 h, 12 h, 14 h, 16 h, 17 h, 18 h or 19 h; the reagent for inducible expression is preferably rhamnose or IPTG; or, the fusion protein has a sequence of SEQ ID NO: 6 or SEQ ID NO: 7; or, the transthyretin is expressed with the protein or polypeptide medicament as a fusion; the fusion is preferably a fusion that the protein or polypeptide medicament are fused at the N-terminus or C-terminus of the transthyretin; preferably, the transthyretin is expressed with the protein or polypeptide medicament as a fusion in a microbial cell, followed by a purification of the fusion; wherein the purification is preferably to remove endotoxin by an endotoxin absorption column and then remove residue bacteria by a filter membrane with a pore size of 0.22 μm; or, the protein or polypeptide medicament comprises lysozyme, albumin or EGFR antibody, with a molecular weight of no more than 45 kDa; the lysozyme is preferably hen egg white lysozyme with GenBank Accession No.: AAL69327.1; the albumin is preferably ovalbumin; or, the protein or polypeptide medicament comprises a protein or polypeptide medicament for treating ocular diseases associated with ocular retina leakage or retinal neovascularization such as diabetic retinopathy, age-related macular degeneration or retinopathy of prematurity.

8. A drops comprising transthyretin or a fusion protein consisting of a transthyretin and a medicament; wherein the medicament is protein or polypeptide medicament, and the transthyretin is represented by (a), (b) or (c): (a) a protein consisting of an amino acid sequence of SEQ ID NO: 1; (b) a protein derived from (a) with an inhibitory function of neovascularization, which is shown by a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (a); (c) a protein having a sequence with a hydrophilic modification or hydrophobic modification in the amino acid sequence of (a) or (b).

9. The drops of claim 8, wherein in (b), the protein derived from (a) is a protein in which 22 amino acids or 25 amino acids are substituted in, or 5 amino acids are deleted from the amino acid sequence of (a); or, in (c), the hydrophilic modification or hydrophobic modification occurs at the cysteine at position 10 of the amino acid sequence of (a).

10. The drops of claim 8, wherein the drops contain the fusion protein consisting of a transthyretin and a medicament, the fusion protein has a content of 4-30 μmol/L, preferably 10-15 μmol/L; or, the transthyretin contained in the drops has a content of 4-30 μmol/L, preferably 5-30 μmol/L, more preferably 10-20 μmol/L, such as 10, 15, 20 μmol/L; or, the drops further comprises saline; or, the drops further comprise a surfactant, wherein the surfactant, for example, is Tween 80, which preferably has a content of 5% (v/v); or, the drops is preferably an eye drops; or, the drops is a drops that inhibits ocular retina leakage or reduces the number of retinal neovascularization; preferably a drops that treats diabetic retinopathy, age-related macular degeneration or retinopathy of prematurity; or, the drops is administered 1-3 times per day, preferably at an amount of 0.3-0.8 nmol protein per eye at each time; or, the drops is administered twice per day, one drop each time, for 3 months; or, the drops is administered once per day, one drop each time, for 5 days; or, the drops is administered twice per day, one drop each time, for 2 weeks; or, the transthyretin is encoded by a nucleotide sequence of SEQ ID NO: 2; or, the transthyretin is expressed by a recombinant expression vector, and the recombinant expression vector has a plasmid backbone comprising a rhamnose inducible promoter, preferably a rhaPBAD promoter; or, the transthyretin is expressed by a recombinant expression vector, the recombinant expression vector has a plasmid backbone of pET-21a or a vector with 25% or more homology therewith, and preferably a vector with 25% or more homology therewith has a sequence of SEQ ID NO: 8; or, the recombinant plasmid expressing transthyretin is encoded by a nucleotide sequence of SEQ ID NO: 3; or, the transthyretin is expressed in a microbial cell, and preferably followed by a purification; the microbial cell is preferably an E. coli, and the E. coli preferably comprises E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5α, E. coli K12 or E. coli TOP10; the purification is preferably to remove endotoxin by an endotoxin absorption column and then remove residue bacteria by a filter membrane with a pore size of 0.22 μm; or, the transthyretin is expressed by culturing the transformant comprising a gene of the transthyretin until the bacteria obtained reaches an OD.sub.600 of 1.5-2.0, such as 1.6, 1.7, 1.8 or 1.9; or, expression of the transthyretin is induced by a reagent for inducible expression, wherein the reagent for inducible expression has a mass volume percentage of 0.1-2%, such as 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.8%, 1.2% or 1.6%, and the expression is preferably induced for a period of 8-20 h, such as 10 h, 12 h, 14 h, 16 h, 17 h, 18 h or 19 h; the reagent for inducible expression is preferably rhamnose or IPTG; or, the fusion protein has a sequence of SEQ ID NO: 6 or SEQ ID NO: 7; or, the transthyretin is expressed with the protein or polypeptide medicament as a fusion; and the fusion is preferably a fusion that the protein or polypeptide medicament are fused at the N-terminus or C-terminus of the transthyretin; preferably, the transthyretin is expressed with the protein or polypeptide medicament as a fusion in a microbial cell, followed by a purification; wherein the purification is preferably to remove endotoxin by an endotoxin absorption column and then remove residue bacteria by a filter membrane with a pore size of 0.22 μm; or, the protein or polypeptide medicament comprises lysozyme, albumin or EGFR antibody, with a molecular weight of no more than 45 kDa; the lysozyme is preferably hen egg white lysozyme with GenBank Accession No.: AAL69327.1; the albumin is preferably ovalbumin; or, the protein or polypeptide medicament comprises a protein or polypeptide medicament for treating ocular diseases associated with ocular retina leakage or retinal neovascularization such as diabetic retinopathy, age-related macular degeneration or retinopathy of prematurity.

11. The drops of claim 8, wherein the drops further comprises a pharmaceutically acceptable excipient which is one or more selected from carboxymethyl cellulose or salts thereof, chondroitin sulfate or salts thereof, dextran, and, hyaluronic acid.

12. The drops of claim 8, wherein the drops further comprises a compound, pharmaceutically acceptable salts thereof, or glycoside thereof; the compound is one or more selected from diclofenac, vitamin A and luteolin.

13. A method for treating ocular diseases associated with ocular retina leakage or retinal neovascularization such as diabetic retinopathy, age-related macular degeneration or retinopathy of prematurity, comprising administering the drops of claim 8 to a patient in need thereof.

14. The method of claim 2, wherein in (b), the protein derived from (a) is a protein in which T3, T5, 126, N27, H31, R34, A36, A37, D38, D39, T40, S50, E61, E63, V65, 168, K70, I73, A81, H90, E92, P102, R104, T123, K126 or E127 are substituted in the amino acid sequence of (a); or a protein in which a deletion occurs at positions 123-127 of the amino acid sequence of (a); wherein, the protein derived from (a) is a protein in which T3G, T5A, I26V, N27D, H31K, R34K, A36T, D39G, T40S, S50A, E61D, E63K, V65T, I68V, K70R, I73L, H90Y, P102H, R104H, T123S, K126Q and E127N are preferably substituted in the amino acid sequence of (a); or a protein in which T3G, T5A, I26V, N27D, H31K, R34K, A36T, A37S, D38E, D39G, T40S, S50A, E61D, E63K, I68V, K70R, I73L, A81T, H90F, E92D, P102H, R104H, T123S, K126Q and E127N are substituted in the amino acid sequence of (a); preferably, the protein derived from (a) has an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11; or, in (c), the hydrophobic modification is a modification with a long chain hydrophobic fragment such as n-dodecane at the cysteine at position 10 of the amino acid sequence of (a); or, the hydrophobic modification is a modification with n-dodecane via maleiamide at the cysteine at position 10 of the amino acid sequence of (a).

15. The method of claim 6, wherein in (b), the protein derived from (a) is a protein in which T3, T5, 126, N27, H31, R34, A36, A37, D38, D39, T40, S50, E61, E63, V65, 168, K70, I73, A81, H90, E92, P102, R104, T123, K126 or E127 are substituted in the amino acid sequence of (a); or a protein in which a deletion occurs at positions 123-127 of the amino acid sequence of (a); preferably, the protein derived from (a) is a protein in which T3G, T5A, I26V, N27D, H31K, R34K, A36T, D39G, T40S, S50A, E61D, E63K, V65T, I68V, K70R, I73L, H90Y, P102H, R104H, T123S, K126Q and E127N are substituted in the amino acid sequence of (a); or a protein in which T3G, T5A, I26V, N27D, H31K, R34K, A36T, A37S, D38E, D39G, T40S, S50A, E61D, E63K, I68V, K70R, I73L, A81T, H90F, E92D, P102H, R104H, T123S, K126Q and E127N are substituted in the amino acid sequence of (a); preferably, the protein derived from (a) has an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11; or, in (c), the hydrophobic modification is a modification with a long chain hydrophobic fragment such as n-dodecane at the cysteine at position 10 of the amino acid sequence of (a); or, the hydrophobic modification is a modification with n-dodecane via maleiamide at the cysteine at position 10 of the amino acid sequence of (a).

16. The drops of claim 9, wherein in (b), the protein derived from (a) is a protein in which T3, T5, 126, N27, H31, R34, A36, A37, D38, D39, T40, S50, E61, E63, V65, 168, K70, I73, A81, H90, E92, P102, R104, T123, K126 or E127 are substituted in the amino acid sequence of (a); or a protein in which a deletion occurs at positions 123-127 of the amino acid sequence of (a); preferably, the protein derived from (a) is a protein in which T3G, T5A, I26V, N27D, H31K, R34K, A36T, D39G, T40S, S50A, E61D, E63K, V65T, I68V, K70R, I73L, H90Y, P102H, R104H, T123S, K126Q and E127N are substituted in the amino acid sequence of (a); or a protein in which T3G, T5A, I26V, N27D, H31K, R34K, A36T, A37S, D38E, D39G, T40S, S50A, E61D, E63K, I68V, K70R, I73L, A81T, H90F, E92D, P102H, R104H, T123S, K126Q and E127N are substituted in the amino acid sequence of (a); preferably, the protein derived from (a) has an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11; or, in (c), the hydrophobic modification is a modification with a long chain hydrophobic fragment such as n-dodecane at the cysteine at position 10 of the amino acid sequence of (a); or, the hydrophobic modification is a modification with n-dodecane via maleiamide at the cysteine at position 10 of the amino acid sequence of (a).

17. The drops of claim 11, wherein the carboxymethyl cellulose or salts thereof has a viscosity of 800-1200 CP; or, the chondroitin sulfate is chondroitin sulfate A; or, the dextran is dextran 70; or, the “salts” in the “carboxymethyl cellulose or salts thereof” or “chondroitin sulfate or salts thereof” is independently sodium salts or calcium salts, such as sodium carboxymethyl cellulose or chondroitin sulfate A sodium salt; or, the hyaluronic acid has a molecular weight of 10000-500000; or, the carboxymethyl cellulose or salts thereof has a concentration of 0-8 mg/mL except 0, preferably 2, 4, 6 or 8 mg/mL; or, the chondroitin sulfate or salts thereof has a concentration of 0-40 mg/mL except 0, preferably 10, 20, 30 or 40 mg/mL; or, the dextran has a concentration of 0-0.8 mg/mL except 0, preferably 0.2, 0.4, 0.6 or 0.8 mg/mL; or, the hyaluronic acid has a content of no more than 6% by mass volume percentage, preferably 1-4%, more preferably 2%.

18. The drops of claim 12, wherein the pharmaceutically acceptable salt is a sodium salt, such as diclofenac sodium; or, the glycoside is luteoloside; or, the vitamin A is vitamin A1 or vitamin A2; or, the diclofenac or salts thereof has a content of 5-20 μmol/L, such as 10 μmol/L; or, the vitamin A has a content of 2-10 μmol/L, such as 5 μmol/L; or, the luteolin or glycoside thereof has a content of 2-10 μmol/L, such as 5 μmol/L.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0273] FIG. 1 shows the three-dimensional structure of transthyretin (TTR) (PDB ID: 1ICT).

[0274] FIG. 2 shows the core hydrophobic domain of TTR carrying strongly hydrophobic thyroxine molecules across various cells.

[0275] FIG. 3 shows that the amino acid sequence similarity among human TTR, TTR derived from SD rats, C57BL/6 mice and rabbits is >95%, wherein 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 mice is shown in SEQ ID NO: 10.

[0276] FIG. 4 shows the plasmid profile of pET-21a (+)-His-tag-TTR-X. The front end of the gene sequence is expressed in fusion with His-tag sequence, and ligated to the plasmid by the two restriction enzyme sites Nde I and EcoR I; “X” represents the protein fused with TTR.

[0277] FIG. 5 shows the electrophoretograms of the expression of human transthyretin in E. coli BL21 (DE3), the purified product of the fusion protein and standards of green fluorescent protein, hen egg white lysozyme and ovalbumin.

[0278] FIG. 6 shows the content of TTR in cornea, vitreous body and fundus oculi samples (retina and choroid) of C57BL/6 mice and SD rats after eye dropping.

[0279] FIG. 7A-D shows the western-blot profile of the target protein in vitreous cavity after eye dropping for 2 weeks with human transthyretin and the fusion protein thereof to rats and rabbits, the left eye is dropping eye, and the right eye is control eye. Since the TTRs derived from human/rats/rabbits have high homology, the vitreous body sample has background TTR positive signal detected by anti-TTR antibody after TTR eye dropping; and the positive signal in the dropping eye increases significantly when detected by anti-His-tag antibody, which illustrates that human TTR can effectively enter the eye and reach the vitreous body; and exogenous protein such as GFP, Lysozyme and Ovalbumin can effectively enter the eye after fusion expression with TTR, while proteins described above cannot enter without fusion expression.

[0280] FIG. 8 shows the retinal leakage and the number of retinal neovascularization of SD rats induced by STZ after eye dropping of TTR.

[0281] FIG. 9 shows that the eye dropping of TTR prevents the modeling process of ROP.

[0282] FIG. 10 shows that the eye dropping of TTR inhibits the pathological process of ROP.

[0283] FIG. 11 shows that the eye dropping of TTR inhibits the pathological process of AMD model.

[0284] FIG. 12 shows the chemical modification process of human TTR.

[0285] FIG. 13A shows that the protein structure is TTR dimer, the diclofenac ligand molecule is represented by arrows, and one molecule of TTR dimer can bind to two molecules of diclofenac.

[0286] FIG. 13B shows the interaction between diclofenac and amino acid residues of TTR.

[0287] FIG. 14A shows that by molecular simulation with Discovery studio software, it is found that vitamin A1 can bind stably to the hydrophobic passage of a TTR polymer, wherein the protein structure is TTR dimer, and vitamin A1 ligand molecule is represented by arrows, one molecule of TTR dimer can bind to one molecule of vitamin A1.

[0288] FIG. 14B shows the interaction between vitamin A1 and amino acid residues of TTR.

[0289] FIG. 15A shows that by molecular simulation with Discovery studio software, it is found that vitamin A2 can bind stably to the hydrophobic passage of a TTR polymer, wherein the protein structure is TTR dimer, vitamin A2 ligand molecule is represented by arrows, and one molecule of TTR dimer can bind to one molecule of vitamin A2.

[0290] FIG. 15B shows the interaction between vitamin A2 and amino acid residues of TTR.

[0291] FIG. 16A shows that by molecular simulation with Discovery studio software, it is found that luteoloside can bind stably to the hydrophobic passage of a TTR polymer, wherein the protein structure is TTR dimer, luteoloside ligand molecule is represented by arrows, and one molecule of TTR dimer can bind to one molecule of luteoloside.

[0292] FIG. 16B shows the interaction between luteoloside and amino acid residues of TTR.

[0293] FIG. 17A shows that by molecular simulation with Discovery studio software, it is found that sulfamethoxazole can bind stably to the hydrophobic passage of a TTR polymer, wherein the protein structure is TTR dimer, sulfamethoxazole ligand molecule is represented by arrows, and one molecule of TTR dimer can bind to one molecule of sulfamethoxazole.

[0294] FIG. 17B shows the interaction between sulfamethoxazole and amino acid residues of TTR.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0295] The following examples further illustrate the present disclosure, but the present disclosure is not limited thereto. In the following examples, the experimental methods without specific conditions are selected according to conventional methods and conditions, or the product specification.

Part I: Preparation of Transthyretin (TTR) and a Fusion Protein Thereof

Example 1 Preparation of Transthyretin

[0296] The preparation of transthyretin comprises the following steps:

[0297] (1) Construction of recombinant plasmid pET-21a (+)-His-tag-TTR: the His-tag-TTR having the nucleotide sequence of SEQ ID NO: 4 (wherein the amino acid sequence of the TTR used is shown in SEQ ID NO: 1, and pET-21a was purchased from ATCC) was synthesized, and ligated to pET-21a (+) with Nde I and EcoR I, followed by verifying a successful construction by sequencing (the sequencing was performed by Nanjing GenScript Biotechnology Ltd., the same below).

[0298] (2) Expression and purification of recombinant TTR: the plasmid pET-21a (+)-His-tag-TTR constructed in step (1) was transformed into E. coli BL21 (DE3) cells, and the recombinant E. coli BL21 (DE3) cells obtained were cultured in LB medium to prepare an inoculum, then inoculating 5% of the inoculum into 5 L of TB medium, incubating at a temperature of 37° C. and paddle speed of 150 rpm until OD.sub.600 of the culture reaches 1.5-2.0; 0.1-0.5 mM IPTG was added to induce for 8-16 h (Table 1). The bacteria was broken by high pressure homogenization, and TTR was prepared by nickel column affinity adsorption of the supernatant thereof. The endotoxin of the protein obtained was removed by an endotoxin absorption column (Pierce™ High Capacity Endotoxin Removal Spin Columns, ThermoFisher) and the residue bacteria was removed by a filter membrane with a pore size of 0.22 μm. The TTR protein yield was determined and the results are shown in Table 1. Wet cells with 17 mg/g and more of protein yield were obtained with induction of 0.3-0.5 mM IPTG for 12-14 h when OD.sub.600 was 1.5-1.8.

TABLE-US-00003 TABLE 1 Expression of TTR under different induction conditions (1) The protein yields were obtained by adding different concentrations of IPTG when the bacteria were cultured to an OD.sub.600 of 1.8 and induced at a temperature of 37° C. for 12 h. IPTG (mM) 0.1 0.2 0.3 0.4 0.5 Protein yield (mg/g wet cells) 5.4 ± 0.6 7.6 ± 0.3 20.8 ± 1.6 19.4 ± 0.9 20.2 ± 1.1 (2) The protein yields were obtained by adding 0.3 mM of IPTG when the bacteria were cultured to different values of OD.sub.600 values and induced at a temperature of 37° C. for 12 h. OD.sub.600 1.5 1.6 1.8 1.9 2.0 Protein yield (mg/g wet cells) 17.4 ± 1.2 19.2 ± 1.5 20.8 ± 1.6 16.3 ± 1.2 14.4 ± 1.3 (3) The protein yields were obtained by adding 0.3 mM of IPTG when the bacteria were cultured to an OD.sub.600 of 1.8 for different times at a temperature of 37° C. Induction time (h) 8 10 12 14 16 Protein yield (mg/g wet cells) 9.3 ± 0.8 11.4 ± 1.2 20.8 ± 1.6 17.3 ± 2.2 15.8 ± 1.7

Example 2: Preparation of Transthyretin-Green Fluorescent Protein Fusion Protein

[0299] The preparation of the fusion protein comprising transthyretin-green fluorescent protein (TTR-GFP) comprises the following steps:

[0300] (1) Construction of recombinant plasmid pET-21a (+)-His-tag-TTR-GFP (the plasmid profile of the following recombinant plasmid pET-21a (+)-His-tag-TTR-X is shown in FIG. 4): the His-tag-TTR-GFP having the nucleotide sequence of SEQ ID NO: 5 was synthesized, and ligated to pET-21a (+) with Nde I and EcoR I, followed by verifying a successful construction by sequencing.

[0301] (2) Expression and purification of TTR-GFP fusion protein: the recombinant plasmid constructed in step (1) was transformed into E. coli BL21 (DE3) cells, and the recombinant E. coli BL21 (DE3) cells obtained were cultured in LB medium to prepare an inoculum, then inoculating 5% of the inoculum into 5 L of TB medium, incubating at a temperature of 37° C. and paddle speed of 150 rpm until OD.sub.600 of the culture reaches 1.5-2.0; 0.1-0.5 mM IPTG was added to induce for 8-16 h. The bacteria was broken by high pressure homogenization, and TTR-GFP fusion protein was prepared by nickel column affinity adsorption of the supernatant thereof. The endotoxin of the protein obtained was removed by an endotoxin absorption column (Pierce™ High Capacity Endotoxin Removal Spin Columns, ThermoFisher) and the residue bacteria was removed by a filter membrane with a pore size of 0.22 The TTR-GFP protein yield was determined and the results are shown in Table 2. Wet cells with 10 mg/g and more of protein yield were obtained with induction of 0.3-0.5 mM IPTG for 12 h when 0D600 was 1.5-1.9.

TABLE-US-00004 TABLE 2 Expression of TTR-GFP under different induction conditions (1) The protein yields were obtained by adding different concentrations of IPTG when the bacteria were cultured to an OD.sub.600 of 1.8 and induced at a temperature of 37° C. for 12 h. IPTG (mM) 0.1 0.2 0.3 0.4 0.5 Protein yield (mg/g wet cells) 3.2 ± 0.1 8.5 ± 0.6 16.4 ± 2.2 14.2 ± 1.4 10.3 ± 0.7 (2) The protein yields were obtained by adding 0.3 mM of IPTG when the bacteria were cultured to different values of OD.sub.600 values and induced at a temperature of 37° C. for 12 h. OD.sub.600 1.5 1.6 1.8 1.9 2.0 Protein yield (mg/g wet cells) 10.2 ± 0.9 14.3 ± 1.1 16.4 ± 2.2 11.6 ± 1.8 9.6 ± 1.0 (3) The protein yields were obtained by adding 0.3 mM of IPTG when the bacteria were cultured to an OD.sub.600 of 1.8 for different times at a temperature of 37° C. Induction time (h) 8 10 12 14 16 Protein yield (mg/g wet cells) 5.3 ± 0.4 7.4 ± 0.5 16.4 ± 2.2 8.6 ± 1.0 6.6 ± 0.5

Example 3: Preparation of Transthyretin-Hen Egg White Lysozyme Fusion Protein

[0302] The preparation of the fusion protein comprising transthyretin-hen egg white lysozyme (TTR-Lysozyme) comprises the following steps:

[0303] (1) Construction of recombinant plasmid pET-21a (+)-His-tag-TTR-Lysozyme: the His-tag-TTR-Lysozyme having the nucleotide sequence of SEQ ID NO: 6 was synthesized, and ligated to pET-21a (+) with Nde I and EcoR I, followed by verifying a successful construction by sequencing.

[0304] (2) Expression and purification of TTR-Lysozyme fusion protein: the recombinant plasmid pET-21a (+)-His-tag-TTR-Lysozyme constructed in step (1) was transformed into E. coli BL21 (DE3) cells, and the recombinant E. coli BL21 (DE3) cells obtained were cultured in LB medium to prepare an inoculum, then inoculating 5% of the inoculum into 5 L of TB medium, incubating at a temperature of 37° C. and paddle speed of 150 rpm until OD.sub.600 of the culture reaches 1.5-2.0; 0.1-0.5 mM IPTG was added to induce for 8-16 h. The bacteria was broken by high pressure homogenization, and TTR-Lysozyme fusion protein was prepared by nickel column affinity adsorption of the supernatant thereof. The endotoxin of the protein obtained was removed by an endotoxin absorption column (Pierce™ High Capacity Endotoxin Removal Spin Columns, ThermoFisher) and the residue bacteria was removed by a filter membrane with a pore size of 0.22 The TTR-Lysozyme protein yield was determined and the results are shown in Table 3.

TABLE-US-00005 TABLE 3 Expression of TTR-Lysozyme under different induction conditions (1) The protein yields were obtained by adding different concentrations of IPTG when the bacteria were cultured to an OD.sub.600 of 1.8 and induced at a temperature of 37° C. for 12 h. IPTG (mM) 0.1 0.2 0.3 0.4 0.5 Protein yield (mg/g wet cells) 8.8 ± 1.3 20.1 ± 1.9 33.4 ± 4.8 21.4 ± 2.6 13.3 ± 2.0 (2) The protein yields were obtained by adding 0.3 mM of IPTG when the bacteria were cultured to different values of OD.sub.600 values and induced at a temperature of 37° C. for 12 h. OD.sub.600 1.5 1.6 1.8 1.9 2.0 Protein yield (mg/g wet cells) 17.3 ± 2.2 24.6 ± 3.1 33.4 ± 4.8 27.2 ± 1.9 14.6 ± 1.8 (3) The protein yields were obtained by adding 0.3 mM of IPTG when the bacteria were cultured to an OD.sub.600 of 1.8 for different times at a temperature of 37° C. Induction time (h) 8 10 12 14 16 Protein yield (mg/g wet cells) 20.2 ± 1.4 28.8 ± 3.4 33.4 ± 4.8 30.3 ± 2.7 25.5 ± 3.0

Example 4: Preparation of Transthyretin-Ovalbumin Fusion Protein

[0305] The preparation of the fusion protein comprising transthyretin-ovalbumin (TTR-Ovalbumin) comprises the following steps:

[0306] (1) Construction of recombinant plasmid pET-21a (+)-His-tag-TTR-Ovalbumin: the His-tag-TTR-Ovalbumin having the nucleotide sequence of SEQ ID NO: 7 was synthesized, and ligated to pET-21a (+) with Nde I and EcoR I, followed by verifying a successful construction by sequencing.

[0307] (2) Expression and purification of TTR-Ovalbumin fusion protein: the recombinant plasmid pET-21a (+)-His-tag-TTR-Ovalbumin constructed in step (1) was transformed into E. coli BL21 (DE3) cells, and the recombinant E. coli BL21 (DE3) cells obtained were cultured in LB medium to prepare an inoculum, then inoculating 5% of the inoculum into 5 L of TB medium, incubating at a temperature of 37° C. and paddle speed of 150 rpm until OD.sub.600 of the culture reaches 1.5-2.0; 0.1-0.5 mM IPTG was added to induce for 8-16 h. The bacteria was broken by high pressure homogenization, and TTR-Ovalbumin fusion protein was prepared by nickel column affinity adsorption of the supernatant thereof. The endotoxin of the protein obtained was removed by an endotoxin absorption column (Pierce™ High Capacity Endotoxin Removal Spin Columns, ThermoFisher) and the residue bacteria was removed by a filter membrane with a pore size of 0.22 The TTR-Ovalbumin protein yield was determined and the results are shown in Table 4.

TABLE-US-00006 TABLE 4 Expression of TTR-Ovalbumin under different induction conditions (1) The protein yields were obtained by adding different concentrations of IPTG when the bacteria were cultured to an OD.sub.600 of 1.8 and induced at a temperature of 37° C. for 12 h. IPTG (mM) 0.1 0.2 0.3 0.4 0.5 Protein yield (mg/g wet cells) 4.2 ± 0.3 6.3 ± 0.4 9.8 ± 0.7 7.3 ± 0.6 7.5 ± 1.2 (2) The protein yields were obtained by adding 0.3 mM of IPTG when the bacteria were cultured to different values of OD.sub.600 values and induced at a temperature of 37° C. for 12 h. OD.sub.600 1.5 1.6 1 . 8 1.9 2.0 Protein yield (mg/g wet cells) 6.7 ± 0.5 8.3 ± 1.1 9.8 ± 0.7 8.4 ± 1.2 7.3 ± 0.6 (3) The protein yields were obtained by adding 0.3 mM of IPTG when the bacteria were cultured to an OD.sub.600 of 1.8 for different times at a temperature of 37° C. Induction time (h) 8 10 12 14 16 Protein yield (mg/g wet cells) 5.5 ± 0.4 8.5 ± 1.0 9.8 ± 0.7 7.4 ± 0.7 5.8 ± 0.8

[0308] FIG. 5 shows the electrophoretograms of the expression of transthyretin in E. coli BL21 (DE3), the purified product of the fusion protein and standards of green fluorescent protein, hen egg white lysozyme and ovalbumin. It can be illustrated that the proteins described above were correctly expressed.

Example 5: Recombinant Preparation of Human Transthyretin

[0309] (1) Construction of recombinant plasmid pETx-rhaPBAD-ttr: plasmid pET-21a (purchased from ATCC) was reformed and reconstructed (the reconstructed plasmid was different from the original plasmid pET-21a at about 75%, and the specific sequence thereof is shown in SEQ ID NO: 8), and T7 promoter was replaced by rhaPBAD promoter (rhamnose inducible), and optimized nucleotide sequence of human TTR (as shown in SEQ ID NO: 2, the amino acid sequence of TTR is shown in SEQ ID NO: 1) was ligated thereto simultaneously. The entire plasmid obtained has a nucleotide sequence of SEQ ID NO: 3. The plasmid was subject to be verified a successful construction by sequencing (the sequencing was performed by Nanjing GenScript Biotechnology Ltd.).

[0310] (2) Expression and purification of recombinant human TTR: the plasmid pETx-rhaPBAD-ttr constructed in step (1) was transformed into E. coli BL21 (DE3) cells, and the recombinant E. coli BL21 (DE3) cells obtained were cultured in LB medium to prepare an inoculum, then inoculating 5% of the inoculum into 5 L of TB medium, incubating at a temperature of 37° C. and paddle speed of 150 rpm until OD.sub.600 of the culture reaches 1.5-2.0; 0.4-2% (mass volume percentage) rhamnose was added to induce for 16-20 h (Table 5). The bacteria was broken by high pressure homogenization, and human TTR was prepared by nickel (Nr) column chromatography of the supernatant thereof. The endotoxin of the protein obtained was removed by an endotoxin absorption column (Pierce™ High Capacity Endotoxin Removal Spin Columns, ThermoFisher) and the residue bacteria was removed by a filter membrane with a pore size of 0.22 μm. The human TTR protein yield was shown in Table 5. Wet cells with 50 mg/g and more of protein yield were obtained with induction of 1.6-2% rhamnose for 18-19 h when OD.sub.600 was 1.8-2.0.

TABLE-US-00007 TABLE 5 Recombinant expression of human TTR (1) The protein yields were obtained by adding different concentrations of rhamnose when the bacteria were cultured to an OD.sub.600 of 1.8 and induced at a temperature of 37° C. for 18 h. Rhamnose (%) 0.4 0.8 1.2 1.6 2.0 Protein yield (mg/g wet cells) 22.3 ± 3.6 32.5 ± 4.3 44.8 ± 5.2 50.9 ± 4.8 51.8 ± 5.3 (2) The protein yields were obtained by adding 1.6% rhamnose when the bacteria were cultured to different values of OD.sub.600 values and induced at a temperature of 37° C. for 18 h. OD.sub.600 1.5 1.6 1.8 1.9 2.0 Protein yield (mg/g wet cells) 33.3 ± 3.2 38.2 ± 4.1 50.9 ± 4.8 53.2 ± 4.8 54.1 ± 5.3 (3) The protein yields were obtained by adding 1.6% rhamnose when the bacteria were cultured to an OD.sub.600 of 1.8 for different times at a temperature of 37° C. Induction time (h) 16 17 18 19 20 Protein yield (mg/g wet cells) 29.9 ± 2.8 37.6 ± 4.2 50.9 ± 4.8 52.2 ± 5.2 45.8 ± 4.7

[0311] (3) The steps for the expression and purification of recombinant rat TTR and mouse TTR are as described above, except that the optimized nucleotide sequence of human TTR was replaced with the nucleotide sequence of corresponding rat/mouse TTR. Wherein, the amino acid sequence of rat TTR is shown in SEQ ID NO: 9, and the amino acid sequence of mouse TTR is shown in SEQ ID NO: 10.

[0312] (4) The protein product of a human mature TTR losing C terminus-TNPKE was obtained according to the steps of recombinant expression described above, and was named human TTR-CL, having the amino acid sequence of SEQ ID NO: 11.

[0313] (5) chemical modification of human TTR: a chemical modification group, which is a hydrophobic modification fragment comprising maleiamide, n-dodecane and 5-aminofluorescein (Ex 490 nm, Em 520 nm), was designed and synthesized, followed by target chemical modification with the only cysteine (C) residue in the human TTR recombinant expressed and purified as described above. Human TTR reacted with the chemical modification group in a molar ratio of 1:5 (the reaction process is shown in FIG. 12) at 4° C. with slow shaking. After the reaction was terminated, the remaining chemical modification agent was discarded by ultrafiltration, and TTR was concentrated. The samples were detected by fluorescence spectrometer with excitation at 490 nm and emission at 520 nm, indicating a successful targeting modification at the unique cysteine residue of TTR, which is named human TTR-Modified.

Example 6: Comparison of Nucleotide Sequences of Human TTR Before and after Optimization

[0314] T7 promoter in the reconstructed plasmid pET-21a as described in Example 5 was replaced with rhaPBAD promoter (rhamnose inducible), followed by ligation to human TTR with an unoptimized nucleotide sequence, thereby obtaining a reconstructed recombinant plasmid pETx-rhaPBAD-ttr (unoptimized), i.e., the only difference between the recombinant plasmid obtained and the recombinant plasmid pETx-rhaPBAD-ttr in Example 5 is whether TTR is optimized. The recombinant human TTR was expressed and purified with the same method described in part (2) of Example 5, and the results are shown in Table 6. It is indicated from the table that TTR proteins are all expressed, and the protein yield of 17 mg/g and more of wet cells can be obtained by inducing with 1.2-2% rhamnose for 17-20 h when OD.sub.600 was 1.6-2.0, indicating a high protein yield.

TABLE-US-00008 TABLE 6 Recombinant expression of human TTR (pETx-rhaPBAD-ttr (unoptimized)) (1) The protein yields were obtained by adding different concentrations of rhamnose when the bacteria were cultured to an OD.sub.600 of 1.8 and induced at a temperature of 37° C. for 18 h. Rhamnose (%) 0.4 0.8 1.2 1.6 2.0 Protein yield (mg/g wet cells) 4.2 ± 0.5 10.2 ± 1.0 17.1 ± 1.1 22.3 ± 2.1 22.5 ± 3.2 (2) The protein yields were obtained by adding 1.6% rhamnose when the bacteria were cultured to different values of OD.sub.600 values and induced at a temperature of 37° C. for 18 h. OD.sub.600 1.5 1.6 1.8 1.9 2.0 Protein yield (mg/g wet cells) 13.2 ± 1.7 18.3 ± 1.6 22.3 ± 2.1 20.4 ± 1.8 17.7 ± 0.9 (3) The protein yields were obtained by adding 1.6% rhamnose when the bacteria were cultured to an OD.sub.600 of 1.8 for different times at a temperature of 37° C. Induction time (h) 16 17 18 19 20 Protein yield (mg/g wet cells) 9.6 ± 1.1 17.8 ± 2.0 22.3 ± 2.1 23.4 ± 3.2 20.4 ± 1.8

[0315] Part II: Transthyretin can Enter Vitreous Cavity and Fundus Oculi Through Corneal Barrier by Itself or Fusing with a Protein

Example 7: Human TTR Enters Vitreous Cavity and Fundus Oculi Through Corneal Barrier in the Manner of Eye Dropping

[0316] (1) The human TTR obtained in Example 5 was prepared as 10 μmol/L (containing saline), and one drop (˜30 μL) was administered by eyedropping to 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 old) respectively. The animals were sacrificed after waiting for 3 h, and the cornea, vitreous body and fundus oculi samples (retina and choroid) were taken out, and the proteins were extracted respectively. Since recombinant human TTR has a His-tag label, rabbit anti-His-tag antibody was used as the primary antibody and donkey anti-rabbit antibody was used as the secondary antibody. Western-blot was conducted to determine whether recombinant human TTR was present in the cornea, vitreous body and fundus oculi samples of C57BL/6 mice and SD rats. The results showed that the signal was not significant in proteins extracted from the corneal tissue, while the signals in vitreous body and fundus oculi samples were significant (FIG. 6), indicating that by the manner of eye dropping, TTR can enter the vitreous body and fundus oculi through the corneal barrier.

[0317] (2) The human TTR obtained in Example 5 was prepared as 5-30 μmol/L (containing saline, and 0-6% hyaluronic acid with low molecular weight), and was administered by eye dropping to C57BL/6 mice (8 weeks old) and SD rats (8 weeks old), respectively. The animals were sacrificed after treatment for 3-72 h, and the proteins were extracted from the vitreous body and fundus oculi samples. Rabbit anti-His-tag antibody was used as the primary antibody, and donkey anti-rabbit antibody was used as the secondary antibody for ELISA to determine the content of human TTR in vitreous body and fundus oculi samples of C57BL/6 mice and SD rats. The results show that when the content of TTR in the eye drops was 10-15 μmol/L, the content of human TTR in the vitreous body and fundus oculi samples reached the peak 3 h after eye dropping; when the concentration of TTR reached 15 μmol/L, further increased concentration of TTR has little effect on increasing the concentration of human TTR in the vitreous body and fundus oculi samples; when the content of hyaluronic acid in the eye drops was 2%, the content of human TTR in the vitreous body and fundus oculi samples reached the peak 3 h after eye dropping; after eye dropping, the content half-life of human TTR in the vitreous body and fundus oculi samples of C57BL/6 mice and SD rats was close to 60 h, indicating that it can effectively exist in the vitreous body and fundus oculi samples for 60 h, with a sufficient therapeutic concentration and therapeutic duration (Table 7-1).

TABLE-US-00009 TABLE 7-1 Human TTR entering the vitreous cavity and fundus oculi through the corneal barrier of C57BL/6 mice and SD rats (1) The human TTR content in vitreous body and fundus oculi 3 h after eye dropping using human recombinant TTR (in saline solution). Concentration of TTR drops (μmol/L) 5 10 15 20 25 30 Concentration of human 0.84 ± 1.81 ± 1.75 ± 1.83 ± 1.88 ± 1.87 ± TTR in vitreous body 0.07 0.19 0.20 0.21 0.17 0.20 and fundus oculi samples of C57BL/6 mice (μmol/L) Concentration of human 1.14 ± 2.43 ± 2.37 ± 2.42 ± 2.45 ± 2.44 ± TTR in vitreous body and 0.11 0.21 0.30 0.36 0.42 0.36 fundus oculi samples of SD rats (μmol/L) (2) The human TTR content in vitreous body and fundus oculi 3 h after eye dropping using human recombinant TTR (10 μmol/L) (in saline solution added with hyaluronic acid of different concentration). Concentration of hyaluronic acid (%) 0 1 2 4 6 Concentration of human 1.81 ± 2.07 ± 2.77 ± 2.13 ± 1.47 ± TTR in vitreous body 0.19 0.16 0.16 0.20 0.11 and fundus oculi samples of C57BL/6 mice (μmol/L) Concentration of human 2.43 ± 2.74 ± 3.11 ± 3.09 ± 3.12 ± TTR in vitreous body and 0.21 0.32 0.31 0.29 0.34 fundus oculi samples of SD rats (μmol/L) (3) The peak of the human TTR content in vitreous body and fundus oculi was reached 3 h after eye dropping using human recombinant TTR (10 μmol/L, comprising saline solution and 2% hyaluronic acid), and the human TTR content thereof at different times thereafter are shown as below Sampling time after eyedropping (h) 3 18 36 60 72 Concentration of human 2.77 ± 2.01 ± 1.73 ± 1.40 ± 0.87 ± TTR in vitreous body 0.16 0.22 0.13 0.11 0.10 and fundus oculi samples of C57BL/6 mice (μmol/L) Concentration of human 3.11 ± 2.74 ± 1.98 ± 1.54 ± 1.02 ± TTR in vitreous body and 0.31 0.26 0.22 0.17 0.06 fundus oculi samples of SD rats (μmol/L)

[0318] (3) The human TTR and human TTR-Modified obtained in Example 5 was prepared as 10 μmol/L (containing saline, and 2% hyaluronic acid with low molecular weight), and was administered by eyedropping to C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) respectively. The animals were sacrificed after treatment for 3-72 h, and the proteins were extracted from the vitreous body and fundus oculi samples (retina and choroid). Rabbit anti-His-tag antibody was used as the primary antibody, and donkey anti-rabbit antibody was used as the secondary antibody for ELISA to determine the content of human TTR in vitreous body and fundus oculi samples of C57BL/6 mice and SD rats. The results show that the efficiency of the human TTR-Modified modified by long hydrophobic fragment entering the vitreous body and fundus oculi is significantly higher than that of unmodified human TTR (Table 7-2).

TABLE-US-00010 TABLE 7-2 Comparison of efficiency of human TTR and human TTR-Modified entering the vitreous cavity and fundus oculi The contents of human TTR and human TTR-Modified in vitreous body and fundus oculi reached the peak 3 h after eyedropping human recombinant TTR and human TTR-Modified (10 μmol/L) (in saline solution added with 2% hyaluronic acid), and the contents thereof at different times thereafter Sampling time after eyedropping (h) 3 18 36 60 72 Concentration of human TTR in vitreous body and 2.77 ± 2.01 ± 1.73 ± 1.40 ± 0.87 ± fundus oculi samples of C57BL/6 mice (μmol/L) 0.16 0.22 0.13 0.11 0.10 Concentration of human TTR-Modified in vitreous 3.62 ± 2.86 ± 2.11 ± 1.89 ± 1.14 ± body and fundus oculi samples of C57BL/6 mice 0.33 0.27 0.19 0.17 0.10 (μmol/L) Concentration of human TTR in vitreous body and 3.11 ± 2.74 ± 1.98 ± 1.54 ± 1.02 ± fundus oculi samples of SD rats (μmol/L) 0.31 0.26 0.22 0.17 0.06 Concentration of human TTR-Modified in vitreous 3.94 ± 3.16 ± 2.44 ± 2.07 ± 1.44 ± body and fundus oculi samples of SD rats (μmol/L) 0.41 0.34 0.24 0.18 0.13

Example 8: TTR Enters the Vitreous Cavity Through the Corneal Barrier by Means of Eye Dropping

[0319] The purified TTR protein (at a concentration of 4 μmol/L) prepared in Example 1 after removal of endotoxin and bacteria was subject to treating healthy SD rats (Rattus norregicus) (6 weeks old) and New Zealand big-eared rabbits (Oryctolagus cuniculus) (2 months old, about 2.5 kg) by means of eye dropping, the left eye was the sample eye dropped with the protein, and the right eye was the blank control eye dropped with saline. The protein was administered 1-3 times per day at a dosage of 0.4-0.8 nmol. Two weeks later, the eyeball was removed and separated to obtain the vitreous body sample. It was preliminarily verified by western-blot method (FIG. 7A) that TTR can enter the vitreous cavity from the ocular surface. The western-blot results with Anti-His tag antibody as the primary antibody show that the signal intensity of the exogenous TTR in the vitreous body of the left eye is 28.7 times that of the right eye in SD rats, and the signal intensity of the exogenous TTR in the vitreous body of the left eye is 35.6 times that of the right eye in New Zealand big-ear rabbits; the content of TTR in the vitreous body sample was determined by ELISA with the anti-His tag antibody as the primary antibody (Table 8 and 9). The results show that after dropping at the content of 0.6 nmol twice a day, higher levels of content of exogenous TTR were detected in the vitreous body of SD rats and New Zealand big-ear rabbits.

[0320] In addition, FIG. 3 shows that the sequence similarity of human/rat/mouse/rabbit-derived transthyretin reaches nearly 95% by homology alignment, indicating that the background positive signal of the TTR in FIG. 7A is intraocular homologous protein signal.

TABLE-US-00011 TABLE 8 Effects of TTR entering the eye of SD rats in different treatment manners (1) Treatment: 0.6 nmol TTR drops in left eye every time, with different times for dropping times for dropping per day 1 2 3 TTR content in vitreous Day 2 23.2 ± 1.8 40.3 ± 5.2 41.1 ± 4.8 body (nmol/L) Day 6 48.9 ± 5.4 69.7 ± 7.4 70.6 ± 7.6 Day 10 61.2 ± 7.3 85.3 ± 7.5 89.2 ± 9.3 Day 14 70.8 ± 6.9 103.2 ± 9.9  107.4 ± 10.1 (2) Treatment: drops twice every day in left eye with different content each time Content for each drops (nmol) 0.4 0.6 0.8 TTR content in vitreous Day 2 20.7 ± 2.3 40.3 ± 5.2 42.8 ± 4.8 body (nmol/L) Day 6 41.5 ± 5.2 69.7 ± 7.4 70.2 ± 5.9 Day 10 57.6 ± 4.9 85.3 ± 7.5 87.9 ± 9.2 Day 14 66.6 ± 7.1 103.2 ± 9.9  107.7 ± 10.3

TABLE-US-00012 TABLE 9 Effects of TTR entering the eye of New Zealand big-ear rabbits in different treatment manners (1) Treatment: 0.6 nmol TTR drops in left eye every time, with different times for dropping times for dropping per day 1 2 3 TTR content in vitreous Day 2 31.5 ± 4.2 49.5 ± 4.5 50.2 ± 4.3 body (nmol/L) Day 6 51.7 ± 6.3 76.3 ± 6.2 76.7 ± 7.1 Day 10 82.6 ± 7.4 97.6 ± 8.7 99.2 ± 8.6 Day 14 89.6 ± 8.8 112.3 ± 9.5  113.2 ± 10.6 (2) Treatment: drops twice every day in left eye with different content each time Content for each drops (nmol) 0.4 0.6 0.8 TTR content in vitreous Day 2 36.6 ± 3.5 49.5 ± 4.5 51.1 ± 4.6 body (nmol/L) Day 6 53.4 ± 5.7 76.3 ± 6.2 79.2 ± 6.3 Day 10 81.6 ± 7.2 97.6 ± 8.7 101.2 ± 9.8  Day 14 89.8 ± 8.3 112.3 ± 9.5  115.4 ± 10.6

Example 9: TTR-GFP Fusion Protein Enters the Vitreous Cavity Through the Corneal Barrier by Means of Eye Dropping

[0321] The purified TTR-GFP protein (at a concentration of 4 μmol/L) prepared in Example 2 after removal of endotoxin and bacteria was subject to treating healthy SD rats (Rattus norregicus) (6 weeks old) and New Zealand big-eared rabbits (Oryctolagus cuniculus) (2 months old, about 2.5 kg) by means of eye dropping, the left eye was the sample eye dropped with the protein, and the right eye was the blank control eye dropped with saline. The protein was administered 1-3 times per day at a dosage of 0.4-0.8 nmol. Two weeks later, the eyeball was removed and separated to obtain the vitreous body sample. It was preliminarily verified by western-blot method (FIG. 7B) that TTR-GFP can enter the vitreous cavity from the ocular surface. The western-blot results with Anti-GFP antibody as the primary antibody show that the signal intensity of the exogenous GFP in the vitreous body of the left eye is 62.3 times that of the right eye in SD rats, and the signal intensity of the exogenous GFP in the vitreous body of the left eye is 47.6 times that of the right eye in New Zealand big-ear rabbits; the western-blot results with Anti-His tag antibody as the primary antibody show that the signal intensity of the exogenous TTR in the vitreous body of the left eye was strong while there was no signal in that of the right eye in SD rats, and the signal intensity of the exogenous TTR in the vitreous body of the left eye is 45.4 times that of the right eye in New Zealand big-ear rabbits. The content of TTR-GFP in the vitreous body sample was determined by ELISA with the anti-His tag antibody as the primary antibody (Table 10 and 11). The results show that after dropping at the content of 0.6 nmol twice a day, higher levels of content of exogenous TTR-GFP were detected in the vitreous body of SD rats and New Zealand big-ear rabbits.

TABLE-US-00013 TABLE 10 Effects of TTR-GFP entering the eye of SD rats in different treatment manners (1) Treatment: 0.6 nmol TTR-GFP drops in left eye every time, with different times for dropping times for dropping per day 1 2 3 TTR-GFP content in Day 2 18.7 ± 1.3 32.2 ± 2.7 33.3 ± 3.1 vitreous body (nmol/L) Day 6 33.2 ± 2.8 51.6 ± 4.9 54.3 ± 6.2 Day 10 40.2 ± 3.6 70.3 ± 6.4 74.2 ± 6.9 Day 14 47.8 ± 5.1 89.9 ± 8.2 91.3 ± 8.7 (2) Treatment: drops twice every day in left eye with different content each time Content for each drops (nmol) 0.4 0.6 0.8 TTR-GFP content in Day 2 11.6 ± 1.7 32.2 ± 2.7 34.6 ± 4.0 vitreous body (nmol/L) Day 6 28.8 ± 3.4 51.6 ± 4.9 54.2 ± 4.9 Day 10 46.7 ± 5.2 70.3 ± 6.4 73.4 ± 7.1 Day 14 55.7 ± 6.1 89.9 ± 8.2  92.2 ± 10.4

TABLE-US-00014 TABLE 11 Effects of TTR-GFP entering the eye of New Zealand big-ear rabbits in different treatment manners (1) Treatment: 0.6 nmol TTR-GFP drops in left eye every time, with different times for dropping times for dropping per day 1 2 3 TTR-GFP content in Day 2 25.3 ± 2.4 42.4 ± 3.8 43.5 ± 4.2 vitreous body (nmol/L) Day 6 40.1 ± 3.8 61.3 ± 5.4 64.1 ± 5.7 Day 10 58.3 ± 4.6 77.6 ± 6.8 79.3 ± 7.5 Day 14 72.2 ± 7.3 90.2 ± 8.7 94.3 ± 8.6 (2) Treatment: drops twice every day in left eye with different content each time Content for each drops (nmol) 0.4 0.6 0.8 TTR-GFP content in Day 2 36.1 ± 3.2 42.4 ± 3.8 44.1 ± 4.5 vitreous body (nmol/L) Day 6 49.9 ± 5.6 61.3 ± 5.4 62.3 ± 5.8 Day 10 57.8 ± 5.7 77.6 ± 6.8 79.2 ± 7.0 Day 14 72.4 ± 6.6 90.2 ± 8.7 91.3 ± 9.2

Example 10: TTR-Lysozyme Fusion Protein Enters the Vitreous Cavity Through the Corneal Barrier by Means of Eye Dropping

[0322] The purified TTR-Lysozyme protein (at a concentration of 4 μmol/L) prepared in Example 3 after removal of endotoxin and bacteria was subject to treating healthy SD rats (Rattus norregicus) (6 weeks old) and New Zealand big-eared rabbits (Oryctolagus cuniculus) (2 months old, about 2.5 kg) by means of eye dropping, the left eye was the sample eye dropped with the protein, and the right eye was the blank control eye dropped with saline. The protein was administered 1-3 times per day at a dosage of 0.4-0.8 nmol. Two weeks later, the eyeball was removed and separated to obtain the vitreous body sample. It was preliminarily verified by western-blot method (FIG. 7C) that TTR-Lysozyme can enter the vitreous cavity from the ocular surface. The western-blot results with Anti-Lysozyme antibody as the primary antibody show that the signal intensity of the exogenous Lysozyme in the vitreous body of the left eye is 34.6 times that of the right eye in SD rats, and the signal intensity of the exogenous Lysozyme in the vitreous body of the left eye is strong while there is no signal in that of the right eye in New Zealand big-ear rabbits; the western-blot results with Anti-His tag antibody as the primary antibody show that the signal intensity of the exogenous TTR in the vitreous body of the left eye was 30.2 times that of the right eye in SD rats, and the signal intensity of the exogenous TTR in the vitreous body of the left eye is 46.3 times that of the right eye in New Zealand big-ear rabbits. The content of TTR-Lysozyme in the vitreous body sample was determined by ELISA with the anti-His tag antibody as the primary antibody (Table 12 and 13). The results show that after dropping at the content of 0.6 nmol twice a day, higher levels of content of exogenous TTR-Lysozyme were detected in the vitreous body of SD rats and New Zealand big-ear rabbits.

TABLE-US-00015 TABLE 12 Effects of TTR-Lysozyme entering the eye of SD rats in different treatment manners (1) Treatment: 0.6 nmol TTR-Lysozyme drops in left eye every time, with different times for dropping times for dropping per day 1 2 3 TTR-Lysozyme content in Day 2 22.1 ± 1.7 37.8 ± 3.2 39.6 ± 4.2 vitreous body (nmol/L) Day 6 39.8 ± 3.4 59.2 ± 5.3 61.7 ± 5.6 Day 10 49.9 ± 5.1 75.6 ± 6.9 80.5 ± 5.9 Day 14 55.2 ± 5.5 96.4 ± 8.8  99.4 ± 10.3 (2) Treatment: drops twice every day in left eye with different content each time Content for each drops (nmol) 0.4 0.6 0.8 TTR-Lysozyme content in Day 2 17.7 ± 1.5 37.8 ± 3.2 39.7 ± 5.0 vitreous body (nmol/L) Day 6 36.5 ± 3.1 59.2 ± 5.3 66.3 ± 5.7 Day 10 50.2 ± 4.8 75.6 ± 6.9 79.9 ± 7.2 Day 14 60.3 ± 5.4 96.4 ± 8.8 100.4 ± 9.6 

TABLE-US-00016 TABLE 13 Effects of TTR-Lysozyme entering the eye of New Zealand big-ear rabbits in different treatment manners (1) Treatment: 0.6 nmol TTR-Lysozyme drops in left eye every time, with different times for dropping times for dropping per day 1 2 3 TTR-Lysozyme content in Day 2 29.7 ± 3.1 49.2 ± 4.6 50.8 ± 5.3 vitreous body (nmol/L) Day 6 48.6 ± 5.3 63.4 ± 5.8 65.4 ± 6.3 Day 10 61.2 ± 5.6 85.7 ± 7.8 87.9 ± 9.0 Day 14 70.4 ± 6.7 107.2 ± 9.6  110.2 ± 10.4 (2) Treatment: drops twice every day in left eye with different content each time Content for each drops (nmol) 0.4 0.6 0.8 TTR-Lysozyme content in Day 2 36.1 ± 3.2 49.2 ± 4.6 51.8 ± 4.7 vitreous body (nmol/L) Day 6 49.4 ± 5.0 63.4 ± 5.8 67.5 ± 6.2 Day 10 70.3 ± 6.5 85.7 ± 7.8 89.9 ± 9.3 Day 14 79.6 ± 6.2 107.2 ± 9.6  110.8 ± 12.1

Example 11: TTR-Ovalbumin Fusion Protein Enters the Vitreous Cavity Through the Corneal Barrier by Means of Eye Dropping

[0323] The purified TTR-Ovalbumin protein (at a concentration of 4 μmol/L) prepared in Example 4 after removal of endotoxin and bacteria was subject to treating healthy SD rats (Rattus norregicus) (6 weeks old) and New Zealand big-eared rabbits (Oryctolagus cuniculus) (2 months old, about 2.5 kg) by means of eye dropping, the left eye was the sample eye dropped with the protein, and the right eye was the blank control eye dropped with saline. The protein was administered 1-3 times per day at a dosage of 0.4-0.8 nmol. Two weeks later, the eyeball was removed and separated to obtain the vitreous body sample. It was preliminarily verified by western-blot method (FIG. 7D) that TTR-Ovalbumin can enter the vitreous cavity from the ocular surface. The western-blot results with Anti-Ovalbumin antibody as the primary antibody show that the signal intensity of the exogenous Ovalbumin in the vitreous body of the left eye is 25.3 times that of the right eye in SD rats, and the signal intensity of the exogenous Ovalbumin in the vitreous body of the left eye is strong while there is no signal in that of the right eye in New Zealand big-ear rabbits; the western-blot results with Anti-His tag antibody as the primary antibody show that the signal intensity of the exogenous TTR in the vitreous body of the left eye is 37.8 times that of the right eye in SD rats, and the signal intensity of the exogenous Ovalbumin in the vitreous body of the left eye is strong while there is no signal in that of the right eye in New Zealand big-ear rabbits. The content of TTR-Ovalbumin in the vitreous body sample was determined by ELISA with the anti-His tag antibody as the primary antibody (Table 14 and 15). The results show that after dropping at the content of 0.6 nmol twice a day, higher levels of content of exogenous TTR-Ovalbumin were detected in the vitreous body of SD rats and New Zealand big-ear rabbits.

TABLE-US-00017 TABLE 14 Effects of TTR-Ovalbumin entering the eye of SD rats in different treatment manners (1) Treatment: 0.6 nmol TTR-Ovalbumin drops in left eye every time, with different times for dropping times for dropping per day 1 2 3 TTR-Ovalbumin content in Day 2 14.4 ± 1.2 27.7 ± 1.9 28.9 ± 3.0 vitreous body (nmol/L) Day 6 24.3 ± 2.1 39.4 ± 3.2 41.3 ± 3.5 Day 10 30.5 ± 2.9 57.8 ± 4.9 60.4 ± 5.7 Day 14 36.7 ± 3.5 70.2 ± 6.4 73.6 ± 8.1 (2) Treatment: drops twice every day in left eye with different content each time Content for each drops (nmol) 0.4 0.6 0.8 TTR-Ovalbumin content in Day 2  9.5 ± 0.7 27.7 ± 1.9 29.2 ± 2.6 vitreous body (nmol/L) Day 6 20.2 ± 1.9 39.4 ± 3.2 43.1 ± 3.0 Day 10 31.7 ± 3.4 57.8 ± 4.9 59.8 ± 6.2 Day 14 43.5 ± 4.1 70.2 ± 6.4 74.5 ± 6.3

TABLE-US-00018 TABLE 15 Effects of TTR-Ovalbumin entering the eye of New Zealand big-ear rabbits in different treatment manners (1) Treatment: 0.6 nmol TTR-Ovalbumin drops in left eye every time, with different times for dropping times for dropping per day 1 2 3 TTR-Ovalbumin content in Day 2 23.9 ± 2.1 31.2 ± 2.8 33.6 ± 3.7 vitreous body (nmol/L) Day 6 30.5 ± 3.2 44.7 ± 5.0 47.2 ± 4.5 Day 10 47.5 ± 4.6 61.3 ± 5.7 65.6 ± 5.9 Day 14 59.9 ± 6.1 75.4 ± 8.0 77.3 ± 8.0 (2) Treatment: drops twice every day in left eye with different content each time Content for each drops (nmol) 0.4 0.6 0.8 TTR-Ovalbumin content in Day 2 19.8 ± 2.4 31.2 ± 2.8 34.3 ± 3.6 vitreous body (nmol/L) Day 6 34.2 ± 2.9 44.7 ± 5.0 46.5 ± 4.5 Day 10 47.6 ± 5.1 61.3 ± 5.7 65.1 ± 6.4 Day 14 58.7 ± 6.2 75.4 ± 8.0 78.7 ± 8.0

Part III: Treating Ocular Diseases Such as DR with TTR

Example 12: Treating DR (Diabetic Retinopathy) SD Rats with Human TTR in the Manner of Eye Dropping

[0324] 8-week-old SD rats weighing 200-250 g was fasted for 12-18 h, and treated with intraperitoneally injection of 2% STZ (60 mg/kg), followed by cutting the tail and collecting blood thereof after 48 h and 72 h. The blood glucose was detected higher than 16.7 mM by test paper, indicating that the model was successfully established, and DR SD rats were obtained. DR SD rats were divided into 2 large groups, one is DR SD rats without any treatment (5 rats), and the other is the human TTR eye dropping group (25 rats) dropped with the human TTR prepared in Example 5, each left eye dropped with human TTR (5-20 μmol/L) (normal saline+2% hyaluronic acid) twice a day, and each right eye dropped with saline+2% hyaluronic acid; in addition, another group of normal SD rats (No eye dropping) served as a normal control group (5 rats). All SD rats were fed for 3 months, followed by stripping the retina for staining with Evans Blue (EB) to observe the retinal vascular leakage, for Trypsin enzymatic hydrolysis to observe the neovascularization density; the results show that compared with the normal control, retinal vascular leakage and the neovascularization number were significantly increased after feeding STZ induced SD rats for 3 months, while the eyeball retinal leakage condition of SD rats treated with human TTR was significantly inhibited, and the number of retinal neovascularization was reduced significantly (FIG. 8), indicating the clinical pathological condition of DR had been alleviated. Wherein, the treatment of administering 10 μmol/L human TTR (saline+2% hyaluronic acid) per day has the best effect (Table 16).

TABLE-US-00019 TABLE 16 DR pathological condition of SD rats induced by STZ treated with human TTR The DR pathological condition of SD rats induced by STZ after 3 months of eyedropping treatment using human recombinant TTR (saline solution + 2% hyaluronic acid) Concentration of 0 5 10 15 20 TTR for eyedropping (μmol/L) Retinal leakage 29.4 ± 3.1 6.2 ± 0.5 5.5 ± 0.4 5.6 ± 0.5 5.3 ± 0.5 area (%) No. of retinal   77 ± 6  14 ± 2  11 ± 1  13 ± 2  12 ± 1 neovascularization (10 visual fields)

Example 13: Treating ROP (Retinopathy of Prematurity) SD Rats with Human TTR in the Manner of Eye Dropping

[0325] One-week-old SD suckling rats were fed in hyperbaric oxygen chamber, and the normal control group was fed in normal environment (normal control group, 6 rats). In hyperbaric oxygen chamber, one group of suckling rats were treated with eye dropping of 5-20 TTR prepared in Example 5 (saline+2% hyaluronic acid), once per day, each drop of 30 μL (ROP/TTR (modeling) group, 6 rats); the other groups of suckling rats was in hyperbaric oxygen chamber without any treatment (ROP group, 24 rats); after fed for 5 days in hyperbaric oxygen chamber, all suckling rats were transferred from hyperbaric oxygen chamber to normal environment for feeding, and one subgroup was separated from ROP group for eye dropping with 5-20 TTR prepared in Example 5 (saline+2% hyaluronic acid), once per day, each drop of 30 μL (ROP/TTR (model) group); other suckling rats in ROP group served as control without any treatment; the suckling rats were sacrificed after feeding for 5 days in normal environment, and the retina was stripped for staining with EB.

[0326] The results are shown in FIG. 9. It is illustrated that in the modeling process, the retina stained with EB of normal control group has normal morphology; ROP group presents obvious no-perfusion area and neovascularization; while modeling, no obvious no-perfusion area and abnormal neovascularization were formed in ROP/TTR (modeling) group treated with eye dropping of TTR, indicating that TTR has protective effect on normal vessels under anoxic condition, and has inhibitory effect on neovascularization (FIG. 9).

[0327] The model ROP suckling rats were treated with eye dropping of TTR (10 μmol/L), 5 days. Compare ROP and ROP/TTR (model) groups, and in the late stage of experiment (5 days), a large number of leakage areas appeared when abnormal neovascularization covered the retina in ROP group, and eye dropping of TTR could reverse this trend (FIG. 10).

[0328] The model ROP suckling rats were treated with eye dropping of different concentrations of TTR (5-20 μmol/L), 5 days. In the late stage of experiment (5 days), a large number of leakage areas appeared when abnormal neovascularization covered the retina in ROP control group, and eye dropping of TTR could reverse this trend, wherein the dosage of 10 μmol/L TTR has the best effect (Table 17).

TABLE-US-00020 TABLE 17 ROP pathological condition of SD suckling rats induced by hyperbaric oxygen chamber treated with human TTR The pathological condition of SD suckling rats induced by hyperbaric oxygen chamber after 5 days of eyedropping treatment using human recombinant TTR (saline solution + 2% hyaluronic acid). Concentration 0 5 10 15 20 of TTR for eyedropping (μmol/L) Retinal leakage 44.7 ± 5.1 9.8 ± 1.0 3.4 ± 0.2 3.2 ± 0.2 3.3 ± 0.2 area (%)

Example 14: Treating AMD (Age-Related Macular Degeneration) C57BL/6 Mice with Human TTR in the Manner of Eye Dropping

[0329] 9-week-old C57BL/6 mice were subject to retinal photocoagulation by krypton laser (647 nm), with a power of 360 mW, a diameter of 50 μm, a time of 0.05 s, 8 photocoagulation site for each eye, for inducing the neovascularization in choroid, and gradually proceeding to retinal hyperplasia to obtain AMD C57BL/6 mice. AMD C57BL/6 mice were divided into a dropping group and a non-dropping group. The dropping group has 14 mice treating with eye dropping of TTR, with the right eye of 5-20 μmol/L TTR prepared in Example 5 (saline+2% hyaluronic acid) twice per day, 30 μL each time, the left eye of saline+2% hyaluronic acid as control; the non-dropping group has 6 mice without any treatment; there was another group (2 mice, without eye dropping) without laser radiation as normal control group; the animals were sacrificed after eye dropping for 2 weeks, and the retina was stripped for EB staining to observe the retinal leakage conditions, and for Trypsin enzymatic hydrolysis to observe the density of neovascularization.

[0330] The results are shown in FIG. 11. It is illustrated that the retina of normal control group has normal morphology in EB staining; AMD group has significant retinal leakage and neovascularization; the retinal leakage conditions and neovascularization conditions of the AMD/TTR group treated with 10 μmol/L TTR have achieved significant alleviation (FIG. 11).

[0331] The modeled AMD mice were treated with eye dropping of different concentrations of TTR (5-20 μmol/L), 2 weeks. More leakage areas appeared, and the number of neovascularization increased significantly in AMD control group, and eye dropping of TTR could reverse this trend, wherein the dosage of 10 μmol/L has the best effect (Table 18).

TABLE-US-00021 TABLE 18 AMD pathological condition of C57BL/6 mice induced by laser retinal photocoagulation treated with human TTR The AMD pathological condition of C57BL/6 mice induced by laser retinal photocoagulation after 2 weeks of eyedropping treatment using human recombinant TTR (saline solution + 2% hyaluronic acid). Concentration 0 5 10 15 20 of TTR for eyedropping (μmol/L) Retinal 24.7 ± 2.2  7.2 ± 0.5  4.5 ± 0.3  4.5 ± 0.2  4.3 ± 0.2 leakage area (%) No. of retinal   80 ± 7   18 ± 2   12 ± 2   13 ± 1   14 ± 3 neovas- cularization (10 visual fields)

[0332] It can be known from the examples described above that the pathological conditions of DR, AMD and ROP in respective animal models can be effectively treated by TTR in the manner of eyedropping.

Example 15: Treating DR (Diabetic Retinopathy) SD Rats with Human TTR/Rat TTR in the Manner of Eye Dropping

[0333] 8-week-old SD rats weighing 200-250 g was fasted for 12-18 h, and treated with intraperitoneally injection of 2% STZ (60 mg/kg), followed by cutting the tail and collecting blood thereof after 48 h and 72 h. The blood glucose was detected higher than 16.7 mM by test paper, indicating that the model was successfully established, and DR SD rats were obtained. DR SD rats were divided into 5 large groups, one group is DR SD rats without any treatment (5 rats), one group is the human TTR eye dropping group (5 rats) dropped with the human TTR prepared in Example 5, each left eye dropped with human TTR (10 μmol/L) (saline+2% hyaluronic acid) twice per day, and each right eye dropped with saline+2% hyaluronic acid; one group is the rat TTR eye dropping group (5 rats) dropped with the rat TTR prepared in Example 5, each left eye dropped with rat TTR (10 μmol/L) (saline+2% hyaluronic acid) twice per day, and each right eye dropped with saline+2% hyaluronic acid; one group is the human TTR-CL eye dropping group (5 rats) dropped with the human TTR-CL prepared in Example 5, each left eye dropped with human TTR-CL (10 μmol/L) (saline+2% hyaluronic acid) twice per day, and each right eye dropped with saline+2% hyaluronic acid; the last one is the human TTR-Modified eye dropping group (5 rats) dropped with the human TTR-Modified prepared in Example 5, each left eye dropped with human TTR-Modified (10 μmol/L) (saline+2% hyaluronic acid) twice per day, and each right eye dropped with saline+2% hyaluronic acid; in addition, another group of normal SD rats (No eye dropping) served as a normal control group (5 rats). All SD rats were fed for 3 months, followed by stripping the retina for staining with Evans Blue (EB) to observe the retinal vascular leakage, for Trypsin enzymatic hydrolysis to observe the density of neovascularization; the results show that compared with the normal control, retinal vascular leakage and the number of neovascularization was significantly increased after feeding STZ induced SD rats for 3 months, while the eyeball retinal leakage conditions of SD rats treated with human TTR, human TTR-CL, human TTR-Modified and rat TTR were significantly inhibited, and the number of retinal neovascularization was reduced significantly, indicating the clinical pathological condition of DR had been alleviated.

TABLE-US-00022 TABLE 19 DR pathological condition of SD rats induced by STZ treated with human TTR/rat TTR The DR pathological condition of SD rats induced by STZ after 3 months of eyedropping treatment using human recombinant TTR/ rat recombinant TTR (saline solution + 2% hyaluronic acid). normal no TTR human human eye dropping human TTR- TTR- rat control control TTR CL Modified TTR Concen- — 0 10 10 10 10 tration of TTR for eyedrop- ping (μmol/L) Retinal 4.9 ± 0.5 29.4 ± 3.1 5.5 ± 0.4 5.7 ± 0.9 10.2 ± 1.3 6.2 ± 0.7 leakage area (%) No. of 11 ± 2  77 ± 6 11 ± 1  10 ± 1  19 ± 4 13 ± 2  retinal neo- vascular- ization (10 visual fields)

Example 16: Treating ROP (Retinopathy of Prematurity) SD Rats with Human TTR/Rat TTR in the Manner of Eye Dropping

[0334] One-week-old SD suckling rats were fed in hyperbaric oxygen chamber, and after 5 days in hyperbaric oxygen chamber, the modeling was successfully completed, then transferred all suckling rats from the hyperbaric oxygen chamber to normal environment for feeding, and some model suckling rats were treated with eye dropping of 10 μmol/L TTR, once per day, 5 days. Compare ROP non-eye dropping group (5 rats), ROP+human TTR eye dropping group (5 rats), ROP+human TTR-CL eye dropping group (5 rats), ROP+human TTR-Modified eye dropping group (5 rats) and ROP+rat TTR eye dropping group (5 rats), and there was another non-ROP model rats (non-eye dropping) as normal control group (5 rats). A large number of leakage areas appeared when abnormal neovascularization covered the retina in ROP group, and eye dropping of TTR could reverse this trend.

TABLE-US-00023 TABLE 20 The ROP pathological condition of SD suckling rats induced by hyperbaric oxygen chamber treated with human TTR/rat TTR The pathological condition of SD suckling rats induced by hyperbaric oxygen chamber after 5 days of eyedropping treatment using human recombinant TTR/rat recombinant TTR (saline solution + 2% hyaluronic acid). normal no TTR human human eye dropping human TTR- TTR- rat control control TTR CL Modified TTR Concentration — 0 10 10 10 10 of TTR for eyedropping (μmol/L) Retinal 3.3 ± 44.7 ± 3.4 ± 3.0 ± 7.8 ± 3.6 ± leakage area 0.3 5.1 0.2 0.1 1.0 0.4 (%)

Example 17: Treating AMD (Age-Related Macular Degeneration) C57BL/6 Mice with Human TTR/Mouse TTR in the Manner of Eye Dropping

[0335] 9-week-old C57BL/6 mice were subject to retinal photocoagulation by krypton laser (647 nm), with a power of 360 mW, a diameter of 50 μm, a time of 0.05 s, 8 photocoagulation site for each eye, for inducing the neovascularization in choroid, and gradually proceeding to retinal hyperplasia to obtain AMD C57BL/6 mice. AMD C57BL/6 mice were divided into a dropping group and a non-dropping group. The dropping group was treated with eye dropping of TTR, with the right eye of 10 μmol/L human TTR or mouse TTR (saline+2% hyaluronic acid) twice per day, 30 μL each time, the left eye of saline+2% hyaluronic acid as control; the dropping groups treated with human TTR, human TTR-CL, human TTR-Modified, or mouse TTR has 5 mice in each group; the non-dropping group has 5 mice without any treatment; another group (5 mice) without laser photocoagulation was served as normal control group (non-dropping). The animals were sacrificed after eye dropping for 2 weeks, and the retina was stripped for EB staining to observe the retinal leakage conditions, and for Trypsin enzymatic hydrolysis to observe the density of neovascularization. It can be known in Table 21 that the eyeball retinal leakage conditions of groups treated with human TTR, human TTR-CL, human TTR-Modified and mouse TTR were inhibited significantly, and the number of retinal neovascularization was reduced significantly, indicating that the clinical pathological conditions of AMD were effectively alleviated.

TABLE-US-00024 TABLE 21 AMD pathological condition of C57BL/6 mice induced by laser retinal photocoagulation treated with human TTR/mouse TTR The AMD pathological condition of C57BL/6 mice induced by laser retinal photocoagulation after 2 weeks of eyedropping treatment using human recombinant TTR/mouse recombinant TTR (saline solution + 2% hyaluronic acid). normal no TTR human human eye dropping human TTR- TTR- mouse control control TTR CL Modified TTR Concen- — 0 10 10 10 10 tration of TTR for eyedrop- ping (μmol/L) Retinal 3.9 ± 0.4 24.7 ± 2.2 4.5 ± 0.3 4.8 ± 0.6 9.4 ± 1.1 4.2 ± 0.5 leakage area (%) No. of 11 ± 3  80 ± 7 12 ± 2  11 ± 2  20 ± 3  10 ± 3  retinal neo- vascular- ization (10 visual fields)

Part IV: Improvement of the Excipient

Example 18: The Use of Sodium Carboxymethyl Cellulose as an Excipient can Promote TTR to Cross the Corneal Barrier

[0336] (1) The human TTR prepared in Example 5 was prepared as 5-30 μmol/L (containing saline), and was administered by eyedropping to C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) respectively. The animals were sacrificed after treatment for 3 h, and the proteins were extracted from the vitreous body and fundus oculi samples. The rabbit anti-His-tag antibody was used as the primary antibody and donkey anti-rabbit antibody was used as the secondary antibody. The content of human TTR in the vitreous body and fundus oculi samples from C57BL/6 mice and SD rats was determined by ELISA.

[0337] (2) The human TTR prepared in Example 5 was prepared as 10 μmol/L (containing saline and 0-8 mg/mL sodium carboxymethyl cellulose (purchased from SinoPharm with a viscosity of 800-1200 CP)), and was administered by eyedropping to C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) respectively. The animals were sacrificed after treatment for 3-72 h, and the proteins were extracted from the vitreous body and fundus oculi samples. The rabbit anti-His-tag antibody was used as the primary antibody and donkey anti-rabbit antibody was used as the secondary antibody. The content of human TTR in the vitreous body and fundus oculi samples from C57BL/6 mice and SD rats was determined by ELISA.

[0338] The results show that the content of human TTR in the vitreous body and fundus oculi samples reached the peak 3 h after eye dropping; when sodium carboxymethyl cellulose was added to the eye drops, the content of human TTR in the vitreous body and fundus oculi samples were both increased significantly (increased more than 10%), and when the content of sodium carboxymethyl cellulose was 6 mg/mL, the content of human TTR in the vitreous body and fundus oculi samples reached the peak 3 h after eye dropping; after eye dropping, the content half-life of human TTR in the vitreous body and fundus oculi samples of C57BL/6 mice and SD rats was close to 72 h, indicating that it can effectively exist in the vitreous body and fundus oculi samples for 72 h, with a sufficient therapeutic concentration and therapeutic duration (Table 22).

TABLE-US-00025 TABLE 22 Human TTR entering the vitreous body and fundus oculi through the corneal barrier of C57BL/6 mice and SD rats (1) The human TTR content in vitreous body and fundus oculi 3 h after eye dropping using human recombinant TTR (in saline solution). Concentration of TTR drops (μmol/L) 5 10 15 20 25 30 Concentration of human TTR 0.84 ± 1.81 ± 1.75 ± 1.83 ± 1.88 ± 1.87 ± in vitreous body and fundus 0.07 0.19 0.20 0.21 0.17 0.20 oculi samples of C57BL/6 mice (μmol/L) Concentration of human TTR 1.14 ± 2.43 ± 2.37 ± 2.42 ± 2.45 ± 2.44 ± in vitreous body and fundus 0.11 0.21 0.30 0.36 0.42 0.36 oculi samples of SD rats (μmol/L) (2) The human TTR content in vitreous body and fundus oculi 3 h after eye dropping using human recombinant TTR (10 μmol/L) (in saline solution added with sodium carboxymethyl cellulose of different concentration). Concentration of sodium carboxymethyl cellulose (mg/mL) 0 2 4 6 8 Concentration of human TTR 1.81 ± 2.01 ± 2.44 ± 2.85 ± 2.80 ± in vitreous body and fundus 0.19 0.17 0.18 0.25 0.17 oculi samples of C57BL/6 mice (μmol/L) Concentration of human TTR 2.43 ± 2.88 ± 3.05 ± 3.24 ± 3.20 ± in vitreous body and fundus 0.21 0.19 0.26 0.29 0.30 oculi samples of SD rats (μmol/L) (3) The peak of human TTR content in vitreous body and fundus oculi was reached 3 h after eyedropping using human recombinant TTR (10 μmol/L) (in saline solution added with 6 mg/mL sodium carboxymethyl cellulose), and the human TTR content thereof a different times thereafter are shown as below Sampling time after eyedropping (h) 3 18 36 60 72 Concentration of human TTR 2.85 ± 2.54 ± 2.14 ± 1.66 ± 1.44 ± in vitreous body and fundus 0.25 0.22 0.10 0.10 0.10 oculi samples of C57BL/6 mice (μmol/L) Concentration of human TTR 3.24 ± 2.96 ± 2.32 ± 1.83 ± 1.63 ± in vitreous body and fundus 0.29 0.27 0.19 0.13 0.15 oculi samples of SD rats (μmol/L)

Example 19: The Use of Dextran 70 as an Excipient can Promote TTR to Cross the Corneal Barrier

[0339] (1) The human TTR prepared in Example 5 was prepared as 5-30 μmol/L (containing saline), and was administered by eyedropping to C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) respectively. The animals were sacrificed after treatment for 3 h, and the proteins were extracted from the vitreous body and fundus oculi samples. The rabbit anti-His-tag antibody was used as the primary antibody and donkey anti-rabbit antibody was used as the secondary antibody. The content of human TTR in the vitreous body and fundus oculi samples from C57BL/6 mice and SD rats was determined by ELISA.

[0340] (2) The human TTR prepared in Example 5 was prepared as 10 μmol/L (containing saline and 0-0.8 mg/mL dextran 70 (purchased from SinoPharm with a molecular weight of 64000-76000)), and was administered by eyedropping to C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) respectively. The animals were sacrificed after treatment for 3-72 h, and the proteins were extracted from the vitreous body and fundus oculi samples. The rabbit anti-His-tag antibody was used as the primary antibody and donkey anti-rabbit antibody was used as the secondary antibody. The content of human TTR in the vitreous body and fundus oculi samples from C57BL/6 mice and SD rats was determined by ELISA.

[0341] The results show that the content of human TTR in the vitreous body and fundus oculi samples reached the peak 3 h after eye dropping; when dextran 70 was added to the eye drops, the content of human TTR in the vitreous body and fundus oculi samples were both increased significantly (increased more than 15%), and when the content of dextran 70 was 0.4 mg/mL, the content of human TTR in the vitreous body and fundus oculi samples reached the peak 3 h after eye dropping; after eye dropping, the content half-life of human TTR in the vitreous body and fundus oculi samples of C57BL/6 mice and SD rats was close to 60 h, indicating that it can effectively exist in the vitreous body and fundus oculi samples for 60 h, with a sufficient therapeutic concentration and therapeutic duration (Table 23).

TABLE-US-00026 TABLE 23 Human TTR entering the vitreous body and fundus oculi through the corneal barrier of C57BL/6 mice and SD rats (1) The human TTR content in vitreous body and fundus oculi 3 h after eye dropping using human recombinant TTR (in saline solution). Concentration of TTR drops (μmol/L) 5 10 15 20 25 30 Concentration of human TTR 0.84 ± 1.81 ± 1.75 ± 1.83 ± 1.88 ± 1.87 ± in vitreous body and fundus 0.07 0.19 0.20 0.21 0.17 0.20 oculi samples of C57BL/6 mice (μmol/L) Concentration of human TTR 1.14 ± 2.43 ± 2.37 ± 2.42 ± 2.45 ± 2.44 ± in vitreous body and fundus 0.11 0.21 0.30 0.36 0.42 0.36 oculi samples of SD rats (μmol/L) (2) The human TTR content in vitreous body and fundus oculi 3 h after eye dropping using human recombinant TTR (10 μmol/L) (in saline solution added with dextran 70 of different concentration). Concentration of dextran 70 (mg/mL) 0 0.2 0.4 0.6 0.8 Concentration of human TTR 1.81 ± 2.47 ± 2.91 ± 2.90 ± 2.87 ± in vitreous body and fundus 0.19 0.20 0.23 0.24 0.21 oculi samples of C57BL/6 mice (μmol/L) Concentration of human TTR 2.43 ± 2.81 ± 3.22 ± 3.18 ± 3.12 ± in vitreous body and fundus 0.21 0.22 0.27 0.30 0.28 oculi samples of SD rats (μmol/L) (3) The peak of human TTR content in vitreous body and fundus oculi was reached 3 h after eyedropping using human recombinant TTR (10 μmol/L) (in saline solution added with 0.4 mg/mL dextran 70), and the human TTR content thereof at different times thereafter are shown as below Sampling time after eyedropping (h) 3 18 36 60 72 Concentration of human TTR 2.91 ± 2.21 ± 1.73 ± 1.48 ± 0.87 ± in vitreous body and fundus 0.23 0.16 0.14 0.13 0.06 oculi samples of C57BL/6 mice (μmol/L) Concentration of human TTR 3.22 ± 2.53 ± 1.86 ± 1.59 ± 0.93 ± in vitreous body and fundus 0.27 0.20 0.11 0.14 0.10 oculi samples of SD rats (μmol/L)

Example 20: The Use of Chondroitin Sulfate A Sodium Salt as an Excipient can Promote TTR to Cross the Corneal Barrier

[0342] (1) The human TTR prepared in Example 5 was prepared as 5-30 μmol/L (containing saline), and was administered by eyedropping to C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) respectively. The animals were sacrificed after treatment for 3 h, and the proteins were extracted from the vitreous body and fundus oculi samples. The rabbit anti-His-tag antibody was used as the primary antibody and donkey anti-rabbit antibody was used as the secondary antibody. The content of human TTR in the vitreous body and fundus oculi samples from C57BL/6 mice and SD rats was determined by ELISA.

[0343] (2) The human TTR prepared in Example 5 was prepared as 10 μmol/L (containing saline and 0-40 mg/mL chondroitin sulfate A sodium salt (purchased from SinoPharm)), and was administered by eyedropping to C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) respectively. The animals were sacrificed after treatment for 3-72 h, and the proteins were extracted from the vitreous body and fundus oculi samples. The rabbit anti-His-tag antibody was used as the primary antibody and donkey anti-rabbit antibody was used as the secondary antibody. The content of human TTR in the vitreous body and fundus oculi samples from C57BL/6 mice and SD rats was determined by ELISA.

[0344] The results show that the content of human TTR in the vitreous body and fundus oculi samples reached the peak 3 h after eye dropping; when chondroitin sulfate A sodium salt was added to the eye drops, the content of human TTR in the vitreous body and fundus oculi samples were both increased significantly (increased more than 17%), and when the content of chondroitin sulfate A sodium salt was 20 mg/mL, the content of human TTR in the vitreous body and fundus oculi samples reached the peak 3 h after eye dropping; after eye dropping, the content half-life of human TTR in the vitreous body and fundus oculi samples of C57BL/6 mice and SD rats was close to 72 h, indicating that it can effectively exist in the vitreous body and fundus oculi samples for 72 h, with a sufficient therapeutic concentration and therapeutic duration (Table 24).

TABLE-US-00027 TABLE 24 Human TTR entering the vitreous body and fundus oculi through the corneal barrier of C57BL/6 mice and SD rats (1) The human TTR content in vitreous body and fundus oculi 3 h after eye dropping using human recombinant TTR (in saline solution). Concentration of TTR drops (μmol/L) 5 10 15 20 25 30 Concentration of human TTR 0.84 ± 1.81 ± 1.75 ± 1.83 ± 1.88 ± 1.87 ± in vitreous body and fundus 0.07 0.19 0.20 0.21 0.17 0.20 oculi samples of C57BL/6 mice (μmol/L) Concentration of human TTR 1.14 ± 2.43 ± 2.37 ± 2.42 ± 2.45 ± 2.44 ± in vitreous body and fundus 0.11 0.21 0.30 0.36 0.42 0.36 oculi samples of SD rats (μmol/L) (2) The human TTR content in vitreous body and fundus oculi 3 h after eye dropping using human recombinant TTR (10 μmol/L) (in saline solution added with chondroitin sulfate A sodium salt of different concentration). Concentration of chondroitin sulfate A sodium salt (mg/mL) 0 10 20 30 40 Concentration of human TTR 1.81 ± 2.57 ± 3.01 ± 2.97 ± 2.84 ± in vitreous body and fundus 0.19 0.22 0.27 0.26 0.26 oculi samples of C57BL/6 mice (μmol/L) Concentration of human TTR 2.43 ± 2.85 ± 3.25 ± 3.20 ± 3.10 ± in vitreous body and fundus 0.21 0.27 0.31 0.30 0.30 oculi samples of SD rats (μmol/L) (3) The peak of human TTR content in vitreous body and fundus oculi was reached 3 h after eyedropping using human recombinant TTR (10 μmol/L) (in saline solution added with 20 mg/mL chondroitin sulfate A sodium salt), and the human TTR content thereof at different times thereafter are shown as below Sampling time after eyedropping (h) 3 18 36 60 72 Concentration of human TTR 3.01 ± 2.66 ± 1.98 ± 1.67 ± 1.48 ± in vitreous body and fundus 0.27 0.27 0.20 0.14 0.11 oculi samples of C57BL/6 mice (μmol/L) Concentration of human TTR 3.25 ± 2.78 ± 2.03 ± 1.83 ± 1.58 ± in vitreous body and fundus 0.31 0.25 0.19 0.17 0.17 oculi samples of SD rats (μmol/L)

Example 21: Treating DR (Diabetic Retinopathy) SD Rats with Human TTR-Excipient in the Manner of Eye Dropping

[0345] 8-week-old SD rats weighing 200-250 g, was fasted for 12-18 h, and treated with intraperitoneally injection of 2% STZ (60 mg/kg), followed by cutting the tail and collecting blood thereof after 48 h and 72 h. The blood glucose was detected higher than 16.7 mM by test paper, indicating that the model was successfully established, and DR SD rats were obtained. DR SD rats were divided into 6 groups, one is DR SD rats without any treatment (5 rats), and the other 5 groups each has 5 rats, and the rats were treated with eye dropping twice per day for the left eye and the right eye, 30 μL each time. Wherein, the left eye was dropped with human TTR prepared in Example 5, or human TTR prepared in Example 5 added with the excipient, specifically with 10 μmol/L human TTR (saline solution), 10 μmol/L human TTR (saline solution+6 mg/mL sodium carboxymethyl cellulose), 10 μmol/L human TTR (saline solution+6 mg/mL PEG400), 10 μmol/L human TTR (saline solution+0.4 mg/mL dextran 70) and 10 μmol/L human TTR (saline solution+20 mg/mL chondroitin sulfate A sodium salt); the right eye was dropped with saline solution, or saline solution added with the excipient as control, specifically with saline solution, saline solution+6 mg/mL sodium carboxymethyl cellulose, saline solution+6 mg/mL PEG400, saline solution+0.4 mg/mL dextran 70 and saline solution+20 mg/mL chondroitin sulfate A sodium salt. In addition, there was another group of normal SD rats as a normal control group (5 rats). All SD rats were fed for 3 months, followed by stripping the retina for staining with Evans Blue (EB) to observe the retinal vascular leakage, for Trypsin enzymatic hydrolysis to observe the density of neovascularization. The results show that compared with the normal control, retinal vascular leakage and the number of neovascularization was significantly increased after feeding STZ induced SD rats for 3 months, while the eyeball retinal leakage condition of SD rats treated with human TTR was significantly inhibited, and the number of retinal neovascularization was reduced significantly, indicating the clinical pathological condition of DR had been alleviated. Wherein, the treatment of administering 10 μmol/L human TTR (saline solution+20 mg/mL chondroitin sulfate A sodium salt) per day has best therapeutic effect (Table 25), while the treatment of administering 10 μmol/L human TTR (saline solution+6 mg/mL PEG400) per day has comparatively poor therapeutic effect.

TABLE-US-00028 TABLE 25 DR pathological condition of SD rats induced by STZ treated with human TTR/human TTR-excipient The DR pathological condition of SD rats induced by STZ after 3 months of eyedropping treatment using human recombinant TTR (saline solution + no excipient/6 mg/mL sodium carboxymethyl cellulose/6 mg/mL PEG400/ 0.4 mg/mL dextran 70/20 mg/mL chondroitin sulfate A sodium salt). human human TTR TTR (saline saline (saline saline human solution + solution + solution + solution + human TTR 20 mg/mL 20 mg/mL human 6 mg/mL 6 mg/mL TTR (saline saline chon- chon- TTR sodium sodium (saline saline solution + solution + droitin droitin normal no TTR (saline saline carboxy- carboxy- solution + solution + 0.4 mg/mL 0.4 mg/mL sulfate A sulfate A control dropping solu- solu- methyl methyl 6 mg/mL 6 mg/mL dextran dextran sodium sodium eye control tion) tion cellulose) cellulose PEG400) PEG400 70) 70 salt) salt Concen- — 0 10 0 10 0 10 0 10 0 10 0 tration of TTR for eyedrop- ping (μmol/L) Retinal 4.9 ± 29.4 ± 6.0 ± 30.8 ± 5.7 ± 30.4 ± 6.7 ± 34.4 ± 5.0 ± 32.8 ± 4.8 ± 28.6 ± leakage 0.5 3.1 0.5 2.5 0.9 3.5 0.5 3.5 0.3 2.9 0.4 3.1 area (%) No. of 11 ± 2 77 ± 6 13 ± 2 80 ± 9 10 ± 1 80 ± 7 14 ± 3 79 ± 7 10 ± 2 75 ± 9 9 ± 1 82 ± 5 retinal neovas- cular- ization (10 visual fields)

Example 22: Treating AMD (Age-Related Macular Degeneration) C57BL/6 Mice with Human TTR-Excipient in the Manner of Eye Dropping

[0346] 9-week-old C57BL/6 mice were subject to retinal photocoagulation by krypton laser (647 nm), with a power of 360 mW, a diameter of 50 μm, a time of 0.05 s, 8 photocoagulation site for each eye, for inducing the neovascularization in choroid, and gradually proceeding to retinal hyperplasia to obtain AMD C57BL/6 mice. AMD C57BL/6 mice were divided into 6 groups, one is AMD C57BL/6 mice without any treatment (5 mice), and the other 5 groups each has 5 mice, and the mice were treated with eye dropping twice per day for the left eye and the right eye, 30 μL each time. Wherein, the left eye was dropped with human TTR prepared in Example 5, or human TTR prepared in Example 5 added with the excipient, specifically with 10 μmol/L human TTR (saline solution), 10 μmol/L human TTR (saline solution+6 mg/mL sodium carboxymethyl cellulose), 10 μmol/L human TTR (saline solution+6 mg/mL PEG400), 10 μmol/L human TTR (saline solution+0.4 mg/mL dextran 70) and 10 μmol/L human TTR (saline solution+20 mg/mL chondroitin sulfate A sodium salt); the right eye was dropped with saline solution, or saline solution added with the excipient as control, specifically with saline solution, saline solution+6 mg/mL sodium carboxymethyl cellulose, saline solution+6 mg/mL PEG400, saline solution+0.4 mg/mL dextran 70 and saline solution+20 mg/mL chondroitin sulfate A sodium salt). In addition, there was another group of normal AMD C57BL/6 mice as a normal control group (5 mice). The animals were sacrificed after eye dropping for 2 weeks, and the retina was stripped for EB staining to observe the retinal leakage conditions, and for Trypsin enzymatic hydrolysis to observe the density of neovascularization.

[0347] The results show that more leakage areas appeared in AMD C57BL/6 mouse control group, and the number of neovascularization increased significantly, while eye dropping of TTR could reverse this trend (Table 26), and TTR represented better therapeutic effect in the groups added with sodium carboxymethyl cellulose, dextran 70 and chondroitin sulfate A sodium salt.

TABLE-US-00029 TABLE 26 AMD pathological condition of C57BL/6 mice induced by laser retinal photocoagulation treated with human TTR/human TTR-excipient The AMD pathological condition of C57BL/6 mice induced by laser retinal photocoagulation after 2 weeks of eyedropping treatment using human recombinant TTR (saline solution + no excipient/6 mg/mL sodium carboxymethyl cellulose/ 6 mg/mL PEG400/0.4 mg/mL dextran 70/20 mg/mL chondroitin sulfate A sodium salt). human human TTR TTR (saline saline (saline saline human solution + solution + solution + solution + human TTR 20 mg/mL 20 mg/mL human 6 mg/mL 6 mg/mL TTR (saline saline chon- chon- TTR sodium sodium (saline saline solution + solution + droitin droitin normal no TTR (saline saline carboxy- carboxy- solution + solution + 0.4 mg/mL 0.4 mg/mL sulfate A sulfate A control dropping solu- solu- methyl methyl 6 mg/mL 6 mg/mL dextran dextran sodium sodium eye control tion) tion cellulose) cellulose PEG400) PEG400 70) 70 salt) salt Concen- — 0 10 0 10 0 10 0 10 0 10 0 tration of TTR for eyedrop- ping (μmol/L) Retinal 3.9 ± 24.7 ± 5.5 ± 26.3 ± 4.0 ± 25.8 ± 4.7 ± 23.9 ± 4.0 ± 29.9 ± 3.7 ± 30.2 ± leakage 0.4 2.2 0.4 3.0 0.2 3.2 0.5 3.0 0.4 2.9 0.3 3.1 area (%) No. of 11 ± 3 80 ± 7 13 ± 2 82 ± 9 11 ± 1 79 ± 8 12 ± 3 83 ± 9 10 ± 1 84 ± 11 10 ± 1 83 ± 8 retinal neovas- cular- ization (10 visual fields)

Example 23: Treating ROP (Retinopathy of Prematurity) SD Rats with Human TTR-Excipient in the Manner of Eye Dropping

[0348] One-week-old SD suckling rats were fed in hyperbaric oxygen chamber, and the normal control group was fed in normal environment (normal control group, 5 rats). The rats were taken out after feeding for 5 days in hyperbaric oxygen chamber to obtain ROP SD rats. All ROP SD rats and rats of normal control group were fed in normal environment for succeeding 5 days. In the 5 days, ROP SD rats were divided into 6 groups, one is ROP SD rats without any treatment (5 rats), and the other 5 groups each has 5 rats, and the rats were treated with eye dropping once per day for the left eye and the right eye, 30 μL each time. Wherein, the left eye was dropped with human TTR prepared in Example 5, or human TTR prepared in Example 5 added with the excipient, specifically with 10 μmol/L human TTR (saline solution), 10 μmol/L human TTR (saline solution+6 mg/mL sodium carboxymethyl cellulose), 10 μmol/L human TTR (saline solution+6 mg/mL PEG400), 10 μmol/L human TTR (saline solution+0.4 mg/mL dextran 70) and 10 μmol/L human TTR (saline solution+20 mg/mL chondroitin sulfate A sodium salt); the right eye was dropped with saline solution, or saline solution added with the excipient as control, specifically with saline solution, saline solution+6 mg/mL sodium carboxymethyl cellulose, saline solution+6 mg/mL PEG400, saline solution+0.4 mg/mL dextran 70 and saline solution+20 mg/mL chondroitin sulfate A sodium salt. The animals were sacrificed after eye dropping for 5 days, and the retina was stripped for EB staining to observe the retinal leakage conditions. A large number of leakage areas appeared when abnormal neovasculars covered the retina in ROP SD rats control group, and eye dropping of TTR could reverse this trend (Table 27), and TTR represented better therapeutic effect in the groups added with sodium carboxymethyl cellulose, dextran 70 and chondroitin sulfate A sodium salt.

TABLE-US-00030 TABLE 27 ROP pathological condition of SD suckling rats induced by hyperbaric oxygen chamber treated with human TTR/human TTR-excipient The ROP pathological condition of SD suckling rats induced by hyperbaric oxygen chamber after 5 days of eyedropping treatment using human recombinant TTR (saline solution + no excipient/6 mg/mL sodium carboxymethyl cellulose/ 6 mg/mL PEG400/0.4 mg/mL dextran 70/20 mg/mL chondroitin sulfate A sodium salt). human human TTR TTR (saline saline (saline saline human solution + solution + solution + solution + human TTR 20 mg/mL 20 mg/mL human 6 mg/mL 6 mg/mL TTR (saline saline chon- chon- TTR sodium sodium (saline saline solution + solution + droitin droitin normal no TTR (saline saline carboxy- carboxy- solution + solution + 0.4 mg/mL 0.4 mg/mL sulfate A sulfate A control dropping solu- solu- methyl methyl 6 mg/mL 6 mg/mL dextran dextran sodium sodium eye control tion) tion cellulose) cellulose PEG400) PEG400 70) 70 salt) salt Concen- — 0 10 0 10 0 10 0 10 0 10 0 tration of TTR for eyedrop- ping (μmol/L) Retinal 3.3 ± 44.7 ± 5.2 ± 46.3 ± 3.2 ± 45.8 ± 4.0 ± 39.7 ± 4.1 ± 48.6 ± 3.0 ± 42.6 ± leakage 0.5 5.1 0.4 6.1 0.2 4.7 0.3 4.0 0.4 5.2 0.2 4.7 area (%)

Part V: Improvement of Ligands

Example 24: In Silicon Simulation of the Binding Forms of TTR and Each Ligand Molecule

[0349] The static co-crystallization model in the PDB database (PDF: 3CFQ) of TTR dimer and diclofenac is shown in FIG. 13A. In FIG. 13A, the protein structure is TTR dimer, the diclofenac ligand molecule is represented by arrows, and one molecule of TTR dimer can bind to two molecules of diclofenac. FIG. 13B shows the interaction between diclofenac and amino acid residues of TTR.

[0350] Through molecular simulation with Discovery studio software, it is found that vitamin A1 (retinol) can bind stably to the hydrophobic passage of a TTR polymer. In FIG. 14A, the protein structure is TTR dimer, vitamin A1 ligand molecule is represented by arrows, and one molecule of TTR dimer can bind to one molecule of vitamin A1. FIG. 14B shows the interaction between vitamin A1 and amino acid residues of TTR.

[0351] Through molecular simulation with Discovery studio software, it is found that vitamin A2 (3-dehydroretinol) can bind stably to the hydrophobic passage of a TTR polymer. In FIG. 15A the protein structure is TTR dimer, vitamin A2 ligand molecule is represented by arrows, and one molecule of TTR dimer can bind to one molecule of vitamin A2. FIG. 15B shows the interaction between vitamin A2 and amino acid residues of TTR.

[0352] Through molecular simulation with Discovery studio software, it is found that luteoloside can bind stably to the hydrophobic passage of a TTR polymer. In FIG. 16A, the protein structure is TTR dimer, luteoloside ligand molecule is represented by arrows, and one molecule of TTR dimer can bind to one molecule of luteoloside. FIG. 16B shows the interaction between luteoloside and amino acid residues of TTR.

[0353] It can be seen from the results described above, the forces between TTR and these ligand molecules are mainly non-covalent forces such as van der Waals forces, hydrogen bonding and hydrophobic interaction, which will not essentially change the chemical structure between TTR and ligands.

Example 25: Determination of Dynamic Specific Binding Parameters of TTR and Each Potential Ligand Molecule

[0354] The affinity binding equilibrium dissociation constant K.sub.d of TTR and each ligand molecule or salts thereof described in Example 24 was determined by nano ITC (TA). In 10 μmol/L TTR solution (1000 μL), 100 μmol/L of each ligand solution described above was dropped at the velocity of 1 μL/min, and the affinity binding equilibrium dissociation constant K.sub.d was calculated using built-in software (Table 28).

TABLE-US-00031 TABLE 28 The affinity binding equilibrium dissociation constant K.sub.d of each ligand molecule and TTR Diclofenac Vitamin A (purchased sodium from SinoPharm, with Vitamin A2 Luteoloside Concentration (purchased Serial No.: (purchased from J (purchased of TTR from CATOCCHM700908, & K Scientific Co., from (10 μmol/L) SinoPharm) CAS: 68-26-8) Ltd., D230075) SinoPharm) Affinity 4.5 × 10.sup.−8 2.27 × 10.sup.−8 2.35 × 10.sup.−8 1.29 × 10.sup.−7 binding equilibrium dissociation constants (mol/L)

[0355] It is shown in Table 28 that the affinity binding equilibrium dissociation constant of each ligand molecule to TTR is nearly 10.sup.−8 mol/L, which is close to the capability of a monoclonal antibody to recognized single antigen epitope, indicating that the ligand molecules described above can recognize and bind TTR specifically.

Example 26: The Complex of Human TTR-Ligand Molecule Enters the Vitreous Body and Fundus Oculi Through the Corneal Barrier

[0356] The human TTR prepared in Example 5 was prepared as 10 μmol/L (containing saline, 2% hyaluronic acid with low molecular weight and 10 μmol/L diclofenac sodium/5 μmol/L vitamin A+5% (v/v) Tween 80/5 μmol/L luteoloside (the addition amount refers to the in silicon simulation results in Example 24)), and was administered by eye dropping to C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) respectively. The animals were sacrificed after treatment for 3-72 h, and the proteins were extracted from the vitreous body and fundus oculi samples. The rabbit anti-His-tag antibody was used as the primary antibody and donkey anti-rabbit antibody was used as the secondary antibody. The content of human TTR in the vitreous body and fundus oculi samples from C57BL/6 mice and SD rats was determined by ELISA. The content of diclofenac sodium in a sample was determined according to the item of diclofenac sodium, page 115, Volume II, Pharmacopoeia of the People's Republic of China (2020); the content of vitamin A in a sample was determined according to the item of vitamin A, page 1472, Volume II, Pharmacopoeia of the People's Republic of China (2020); the content of luteoloside in a sample was determined according to the item of Luteolin-7-glucoside, British Pharmacopoeia BP2017.

[0357] After eye dropping, the half-life of human TTR and each ligand molecule content in the vitreous body and fundus oculi samples of C57BL/6 mice and SD rats was close to 60 h, indicating that they can effectively exist in the vitreous body and fundus oculi samples for 60 h, with a sufficient therapeutic concentration and therapeutic duration (Table 29).

TABLE-US-00032 TABLE 29 Human TTR and ligand molecules entering the vitreous body and fundus oculi through the corneal barrier of C57BL/6 mice and SD rats The peaks of human TTR and diclofenac sodium contents in vitreous body and fundus oculi were reached 3 h after eye dropping using human recombinant TTR (10 μmol/L) (in saline solution added with 2% hyaluronic acid and 10 μmol/L) diclofenac sodium), and the human TTR and diclofenac sodium contents thereof at different times thereafter are shown as below Sampling time after eye dropping (h) 3 18 36 60 72 Concentration of human TTR in vitreous body and 2.72 ± 1.97 ± 1.69 ± 1.37 ± 0.85 ± fundus oculi samples of C57BL/6 mice (μmol/L) 0.21 0.16 0.14 0.10 0.10 Concentration of diclofenac sodium in vitreous body 2.54 ± 1.81 ± 1.55 ± 1.28 ± 0.81 ± and fundus oculi samples of C57BL/6 mice (μmol/L) 0.17 0.13 0.10 0.10 0.10 Concentration of human TTR in vitreous body and 3.22 ± 2.81 ± 1.93 ± 1.65 ± 0.93 ± fundus oculi samples of SD rats (μmol/L) 0.25 0.28 0.20 0.15 0.07 Concentration of diclofenac sodium in vitreous body 3.01 ± 2.66 ± 1.74 ± 1.48 ± 0.92 ± and fundus oculi samples of SD rats (μmol/L) 0.19 0.21 0.15 0.10 0.08 The peaks of human TTR and vitamin A contents in vitreous body and fundus oculi were reached 3 h after eye dropping using human recombinant TTR (10 μmol/L) (in saline solution added with 2% hyaluronic acid, 5 μmol/L vitamin A and 5% Tween 80), and the human TTR and vitamin A contents thereof at different times thereafter are shown as below Sampling time after eye dropping (h) 3 18 36 60 72 Concentration of human TTR in vitreous body and 2.83 ± 2.04 ± 1.77 ± 1.44 ± 1.02 ± fundus oculi samples of C57BL/6 mice (μmol/L) 0.30 0.18 0.15 0.13 0.10 Concentration of vitamin A in vitreous body and 1.52 ± 0.96 ± 0.85 ± 0.73 ± 0.60 ± fundus oculi samples of C57BL/6 mice (μmol/L) 0.11 0.10 0.09 0.07 0.04 Concentration of human TTR in vitreous body and 3.34 ± 2.99 ± 2.06 ± 1.70 ± 1.23 ± fundus oculi samples of SD rats (μmol/L) 0.27 0.30 0.22 0.14 0.10 Concentration of vitamin A in vitreous body and 1.68 ± 1.53 ± 1.02 ± 0.88 ± 0.65 ± fundus oculi samples of SD rats (μmol/L) 0.11 0.10 0.10 0.06 0.06 The peaks of human TTR and luteoloside contents in vitreous body and fundus oculi were reached 3 h after eye dropping using human recombinant TTR (10 μmol/L) (in saline solution added with 2% hyaluronic acid and 5 μmol/L luteoloside), and the human TTR and luteoloside contents thereof at different times thereafter are shown as below Sampling time after eye dropping (h) 3 18 36 60 72 Concentration of human TTR in vitreous body and 2.56 ± 1.97 ± 1.54 ± 1.30 ± 0.90 ± fundus oculi samples of C57BL/6 mice (μmol/L) 0.22 0.20 0.13 0.10 0.06 Concentration of luteoloside in vitreous body and 1.20 ± 0.92 ± 0.72 ± 0.60 ± 0.40 ± fundus oculi samples of C57BL/6 mice (μmol/L) 0.10 0.08 0.05 0.06 0.00 Concentration of human TTR in vitreous body and 3.12 ± 2.80 ± 2.01 ± 1.57 ± 1.01 ± fundus oculi samples of SD rats (μmol/L) 0.25 0.25 0.20 0.10 0.05 Concentration of luteoloside in vitreous body and 1.48 ± 1.35 ± 0.96 ± 0.71 ± 0.52 ± fundus oculi samples of SD rats (μmol/L) 0.10 0.10 0.05 0.05 0.00

Example 27: Treating DR (Diabetic Retinopathy) SD Rats with Human TTR-Ligand Molecule Complex in the Manner of Eye Dropping

[0358] 8-week-old SD rats weighing 200-250 g, was fasted for 12-18 h, and treated with intraperitoneally injection of 2% STZ (60 mg/kg), followed by cutting the tail and collecting blood thereof after 48 h and 72 h. The blood glucose was detected higher than 16.7 mM by test paper, indicating that the model was successfully established, and DR SD rats were obtained. DR SD rats were divided into 5 groups, one is DR SD rats without any treatment (5 rats), and the other 4 groups each has 5 rats, and the rats were treated with eye dropping twice per day for the left eye and the right eye, 30 μL each time. Wherein, the left eye was dropped with human TTR prepared in Example 5, or human TTR prepared in Example 5 added with the ligand, specifically with 10 μmol/L human TTR (saline solution+2% hyaluronic acid), 10 μmol/L human TTR (saline solution+2% hyaluronic acid+10 diclofenac sodium), 10 μmol/L human TTR (saline solution+2% hyaluronic acid+5 μmol/L vitamin A+5% Tween 80) and 10 μmol/L human TTR (saline solution+2% hyaluronic acid+5 luteoloside); the right eye was dropped with saline solution+2% hyaluronic acid, or saline solution+2% hyaluronic acid+ligand as control, specifically with saline solution+2% hyaluronic acid, saline solution+2% hyaluronic acid+10 diclofenac sodium, saline solution+2% hyaluronic acid+5 μmol/L vitamin A+5% Tween 80 and saline solution+2% hyaluronic acid+5 μmol/L luteoloside. In addition, there was another group of normal SD rats as a normal control group (5 rats). All SD rats were fed for 3 months, followed by stripping the retina for staining with Evans Blue (EB) to observe the retinal vascular leakage, for Trypsin enzymatic hydrolysis to observe the density of neovascularization.

[0359] The results show that compared with the normal control, retinal vascular leakage and the number of neovascularization were significantly increased in DR SD rats control group without any treatment after feeding STZ induced SD rats for 3 months, while the eyeball retinal leakage condition of groups treated with human TTR or human TTR/diclofenac sodium, human TTR/vitamin A, human TTR/luteoloside was significantly inhibited, and the number of retinal neovascularization was reduced significantly, indicating the clinical pathological condition of DR had been alleviated (Table 30).

TABLE-US-00033 TABLE 30 DR pathological condition of SD rats induced by STZ treated with human TTR/human TTR-ligand molecule complex The DR pathological condition of SD rats induced by STZ after 3 months of eye dropping treatment using human recombinant TTR (in saline solution added with 2% hyaluronic acid, and no ligand/10 μmol/L diclofenac sodium/5 μmol/L vitamin A + 5% Tween 80/5 μmol/L luteoloside). human human normal no TTR human TTR/ TTR/ human control dropping TTR diclofenac vitamin TTR/ eye control (no ligand) sodium A luteoloside Concentration of TTR — 0 10 10 10 10 for eye dropping (μmol/L) Retinal leakage 4.9 ± 0.5 29.4 ± 3.1  5.5 ± 0.4 2.2 ± 0.1 3.2 ± 0.2 4.5 ± 0.3 area (%) No. of retinal 11 ± 2  77 ± 6  11 ± 1  7 ± 2 7 ± 3 9 ± 2 neovascularization (10 visual fields)

Example 28: Treating AMD (Age-Related Macular Degeneration) C57BL/6 Mice with Human TTR-Excipient in the Manner of Eye Dropping

[0360] 9-week-old C57BL/6 mice were subject to retinal photocoagulation by krypton laser (647 nm), with a power of 360 mW, a diameter of 50 μm, a time of 0.05 s, 8 photocoagulation site for each eye, for inducing the neovascularization in choroid, and gradually proceeding to retinal hyperplasia to obtain AMD C57BL/6 mice. AMD C57BL/6 mice were divided into 5 groups, one is AMD C57BL/6 mice without any treatment (5 mice), and the other 4 groups each has 5 mice, and the mice were treated with eye dropping twice per day for the left eye and the right eye, 30 μL each time. Wherein, the left eye was dropped with human TTR prepared in Example 5, or human TTR prepared in Example 5 added with the ligand, specifically with 10 μmol/L human TTR (saline solution+2% hyaluronic acid), 10 μmol/L human TTR (saline solution+2% hyaluronic acid+10 diclofenac sodium), 10 μmol/L human TTR (saline solution+2% hyaluronic acid+5 μmol/L vitamin A+5% Tween 80) and 10 μmol/L human TTR (saline solution+2% hyaluronic acid+5 μmol/L luteoloside); the right eye was dropped with saline solution+2% hyaluronic acid, or saline solution+2% hyaluronic acid+ligand as control, specifically with saline solution+2% hyaluronic acid, saline solution+2% hyaluronic acid+10 diclofenac sodium, saline solution+2% hyaluronic acid+5 μmol/L vitamin A+5% Tween 80 and saline solution+2% hyaluronic acid+5 luteoloside. In addition, there was another group of normal C57BL/6 mice as a normal control group (5 mice). The animals were sacrificed after eye dropping for 2 weeks, and the retina was stripped for EB staining to observe the retinal leakage conditions, and for Trypsin enzymatic hydrolysis to observe the density of neovascularization.

[0361] The results are shown in Table 31. In Table 31, it shows that compared with the normal control, retinal vascular leakage and the number of neovascularization were significantly increased in AMD C57BL/6 mice control group without any treatment, while the eyeball retinal leakage condition and neovascularization condition of groups treated with human TTR or human TTR/diclofenac sodium, human TTR/vitamin A, human TTR/luteoloside were significantly alleviated.

TABLE-US-00034 TABLE 31 AMD pathological condition of C57BL/6 mice induced by laser retinal photocoagulation treated with human TTR/human TTR-ligand molecule complex The AMD pathological condition of C57BL/6 mice induced by laser retinal photocoagulation after 2 weeks of eye dropping treatment using human recombinant TTR (in saline solution added with 2% hyaluronic acid, and no ligand/10 μmol/L diclofenac sodium/5 μmol/L vitamin A + 5% Tween 80/5 μmol/L luteoloside). normal no TTR human human human human control dropping TTR TTR/diclofenac TTR/vitamin TTR/ eye control (no ligand) sodium A luteoloside Concentration of — 0 10 10 10 10 TTR for eye dropping (μmol/L) Retinal leakage area 3.9 ± 0.4 24.7 ± 2.2  4.5 ± 0.3 2.8 ± 0.1 3.2 ± 0.1  4.4 ± 0.35 (%) No. of retinal 11 ± 3  80 ± 7  12 ± 2  6 ± 3 7 ± 2 12 ± 1  neovascularization (10 visual fields)

Example 29: Treating ROP (Retinopathy of Prematurity) SD Rats with Human TTR-Ligand Molecule Complex in the Manner of Eye Dropping

[0362] One-week-old SD suckling rats were fed in hyperbaric oxygen chamber, and the normal control group was fed in normal environment (normal control group, 5 rats). The rats were taken out after feeding for 5 days in hyperbaric oxygen chamber to obtain ROP SD rats. All ROP SD rats and rats of normal control group were fed in normal environment for succeeding 5 days. In the 5 days, ROP SD rats were divided into 5 groups, one is ROP SD rats without any treatment (5 rats), and the other 4 groups each has 5 rats, and the rats were treated with eye dropping once per day for the left eye and the right eye, 30 μL each time. Wherein, the left eye was dropped with human TTR prepared in Example 5, or human TTR prepared in Example 5 added with the ligand, specifically with 10 μmol/L human TTR (saline solution+2% hyaluronic acid), 10 μmol/L human TTR (saline solution+2% hyaluronic acid+10 μmol/L diclofenac sodium), 10 μmol/L human TTR (saline solution+2% hyaluronic acid+5 μmol/L vitamin A+5% Tween 80) and 10 μmol/L human TTR (saline solution+2% hyaluronic acid+5 μmol/L luteoloside); the right eye was dropped with saline solution+2% hyaluronic acid, or saline solution+2% hyaluronic acid+ligand as control, specifically with saline solution+2% hyaluronic acid, saline solution+2% hyaluronic acid+10 μmol/L diclofenac sodium, saline solution+2% hyaluronic acid+5 μmol/L vitamin A+5% Tween 80 and saline solution+2% hyaluronic acid+5 μmol/L luteoloside. The animals were sacrificed after eye dropping for 5 days, and the retina was stripped for EB staining to observe the retinal leakage conditions.

[0363] The results are shown in Table 32. It is shown in the table that compared with the normal control, retinal vascular leakage were significantly increased in ROP SD rats control group without any treatment, while the eyeball retinal leakage condition of groups treated with human TTR or human TTR/diclofenac sodium, human TTR/vitamin A, human TTR/luteoloside were significantly alleviated.

TABLE-US-00035 TABLE 32 ROP pathological condition of SD suckling rats induced by hyperbaric oxygen chamber treated with human TTR/human TTR-ligand molecule complex The ROP pathological condition of SD suckling rats induced by hyperbaric oxygen chamber after 5 days of eye dropping treatment using human recombinant TTR (in saline solution added with 2% hyaluronic acid, and no ligand/10 μmol/L diclofenac sodium/5 μmol/L vitamin A + 5% Tween 80/5 μmol/L luteoloside). no TTR human human human human normal dropping TTR TTR/diclofenac TTR/vitamin TTR/ control eye control (no ligand) sodium A luteoloside Concentration — 0 10 10 10 10 of TTR for eye dropping (μmol/L) Retinal leakage 3.3 ± 0.3 44.7 ± 5.1 3.4 ± 0.2 2.0 ± 0.1 2.3 ± 0.1 4.0 ± 0.3 area (%)

[0364] Combining the data of Examples 27-29, it can be seen that even in normal SD rats, the retinal leakage phenomenon is more severe in adult SD rats than that in juvenile SD rats. In addition, other causes such as experimental operations and individual difference, also can lead to retinal leakage and increased number of neovascularization, which indicates that even normal control eyes without modeling have a certain degree of retinal leakage and increased neovascularization. By using human TTR/diclofenac sodium or human TTR/vitamin A, it can not only treat DR, AMD and ROP, but also have the effect of nourishment, which has certain alleviation effect on retinal leakage and increased neovascularization caused by other reasons in an eye itself.

Comparative Example 1: Human TTR is Recombinant Expressed with Different Plasmids

[0365] (1) Construction of recombinant plasmids pET-28a-ttr, pQE-30-ttr and pQE-60-ttr: the optimized DNA sequence (as shown in SEQ ID NO: 2) of mature TTR was ligated to plasmid pET-28a (pET-28a, pQE-30 and pQE-60 all purchased from ATCC) by two restriction enzyme sites Nde I and Hind III to construct plasmid pET-28a-ttr; the optimized DNA sequence (as shown in SEQ ID NO: 2) of mature TTR was ligated to plasmid pQE-30 by two restriction enzyme sites BamH I and Hind III to construct plasmid pQE-30-ttr; the optimized DNA sequence (as shown in SEQ ID NO: 2) of mature TTR was ligated to plasmid pQE-60 by two restriction enzyme sites EcoR I and Hind III to construct plasmid pQE-60-ttr. The plasmids were subject to be verified successful constructions by sequencing (the sequencing was performed by Nanjing GenScript Biotechnology Ltd.).

[0366] (2) Expression and purification of recombinant human TTR: plasmids pET-28a-ttr, pQE-30-ttr and pQE-60-ttr constructed in step (1) and pETx-rhaPBAD-ttr constructed in Example 5 were transformed into E. coli BL21 (DE3) cells, and the recombinant E. coli BL21 (DE3) cells obtained were cultured in LB medium to prepare an inoculum, then inoculating 5% of the inoculum into 5 L of TB medium, incubating at a temperature of 37° C. and paddle speed of 150 rpm until OD.sub.600 of the culture reaches 1.5-2.0; 0.2 mM IPTG was added into the former three media to induce for 18 h, and 1.6% rhamnose was added into the last medium to induce for 18 h. The bacteria was broken by high pressure homogenization, and human TTR was prepared by Ni+ column chromatography of the supernatant thereof. The endotoxin of the protein obtained was removed by an endotoxin absorption column (Pierce™ High Capacity Endotoxin Removal Spin Columns, ThermoFisher) and the residue bacteria was removed by a filter membrane with a pore size of 0.22 The human TTR protein yield was shown in Table 33.

TABLE-US-00036 TABLE 33 Human TTR is recombinant expressed with different plasmids The protein yields were obtained by adding 0.2 mM IPTG or 1.6% rhamnose when the bacteria were cultured to an OD.sub.600 of 1.8 and induced at a temperature of 37° C. for 18 h. Plasmid pET-28a-ttr pQE-30-ttr pQE-60-ttr pETx-rhaPBAD-ttr Protein no soluble no soluble no soluble 50.9 ± 4.8 yield (mg/g expression expression expression wet cells)

[0367] It can be known from the results described above that, when the protein is expressed using pET-28a, pQE-30, pQE-60, etc., insoluble expression occurred.

Comparative Example 2

[0368] Compare with Table 7-1 in Example 7, when the concentration of TTR for eye dropping is 1 μmol/L, the concentration of TTR in the vitreous body and fundus oculi is only 0.10±0.00 μmol/L.

Comparative Example 3

[0369] The detailed operation manner is the same with Example 9 except the difference that, GFP protein (Genbank Accession No.: QAA95705.1) not fused with TTR was expressed according to the method described in Example 2, followed by eye dropping test on SD rats and New Zealand big-ear rabbits according to the operation steps which are the same with Example 9. The results show that GFP did not enter the vitreous body of SD rats or New Zealand big-ear rabbits (Table 34 and 35).

TABLE-US-00037 TABLE 34 Effects of GFP entering the eye of SD rats with different times of drops at the GFP dosage of 0.6 nmol Times of dropping per day 1 2 3 GFP content in vitreous day 2 — — — body (nmol/L) day 6 — — — day 10 — — — day 14 — — —

[0370] Wherein, “−” represents undetected (the same below).

TABLE-US-00038 TABLE 35 Effects of GFP entering the eye of New Zealand big-ear rabbits with different times of drops at the GFP dosage of 0.6 nmol Times of dropping per day 1 2 3 GFP content in vitreous day 2 — — — body (nmol/L) day 6 — — — day 10 — — — day 14 — — —

Comparative Example 4

[0371] The detailed operation manner is the same with Example 10 except the difference that, Lysozyme protein (Genbank Accession No.: AAL69327.1) not fused with TTR was expressed according to the method described in Example 3, followed by eye dropping test on SD rats and New Zealand big-ear rabbits according to the operation steps which are the same with Example 10. The results show that Lysozyme did not enter the vitreous body of SD rats or New Zealand big-ear rabbits (Table 36 and 37).

TABLE-US-00039 TABLE 36 Effects of Lysozyme entering the eye of SD rats with different times of drops at the Lysozyme dosage of 0.6 nmol Times of dropping per day 1 2 3 Lysozyme content in day 2 — — — vitreous body (nmol/L) day 6 — — — day 10 — — — day 14 — — —

TABLE-US-00040 TABLE 37 Effects of Lysozyme entering the eye of New Zealand big-ear rabbits with different times of drops at the Lysozyme dosage of 0.6 nmol Times of dropping per day 1 2 3 Lysozyme content in day 2 — — — vitreous body (nmol/L) day 6 — — — day 10 — — — day 14 — — —

Comparative Example 5

[0372] The detailed operation manner is the same with Example 11 except the difference that, Ovalbumin protein (UniProt Accession No.: P01012) not fused with TTR was expressed according to the method described in Example 4, followed by eye dropping test on SD rats and New Zealand big-ear rabbits according to the operation steps which are the same with Example 11. The results show that Ovalbumin did not enter the vitreous body of SD rats or New Zealand big-ear rabbits (Table 38 and 39).

TABLE-US-00041 TABLE 38 Effects of Ovalbumin entering the eye of SD rats with different times of drops at the Ovalbumin dosage of 0.6 nmol Times of dropping per day 1 2 3 Ovalbumin content in day 2 — — — vitreous body (nmol/L) day 6 — — — day 10 — — — day 14 — — —

TABLE-US-00042 TABLE 39 Effects of Ovalbumin entering the eye of New Zealand big-ear rabbits with different times of drops at the Ovalbumin dosage of 0.6 nmol Times of dropping per day 1 2 3 Ovalbumin content in day 2 — — — vitreous body (nmol/L) day 6 — — — day 10 — — — day 14 — — —

Comparative Example 6

[0373] (1) The human TTR prepared in Example 5 was prepared as 5-30 μmol/L (containing saline), and was administered by eye dropping to C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) respectively. The animals were sacrificed after treatment for 3 h, and the proteins were extracted from the vitreous body and fundus oculi samples. The rabbit anti-His-tag antibody was used as the primary antibody and donkey anti-rabbit antibody was used as the secondary antibody. The content of human TTR in the vitreous body and fundus oculi samples from C57BL/6 mice and SD rats was determined by ELISA.

[0374] (2) The human TTR prepared in Example 5 was prepared as 10 μmol/L (containing saline and 0-8 mg/mL PEG400 (purchased from SinoPharm, molecular weight of 360-440)), and was administered by eye dropping to C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) respectively. The animals were sacrificed after treatment for 3-72 h, and the proteins were extracted from the vitreous body and fundus oculi samples. The rabbit anti-His-tag antibody was used as the primary antibody and donkey anti-rabbit antibody was used as the secondary antibody. The content of human TTR in the vitreous body and fundus oculi samples from C57BL/6 mice and SD rats was determined by ELISA.

[0375] The results show that the content of human TTR in the vitreous body and fundus oculi samples reached the peak 3 h after eye dropping; when PEG400 was added to the eye drops, the content of human TTR in the vitreous body and fundus oculi samples were both increased by more than 20%, and when the content of PEG400 was 6 mg/mL, the content of human TTR in the vitreous body and fundus oculi samples reached the peak 3 h after eye dropping; after eye dropping, the content half-life of human TTR in the vitreous body and fundus oculi samples of C57BL/6 mice and SD rats was close to 60 h, indicating that it can effectively exist in the vitreous body and fundus oculi samples for 60 h, with a sufficient therapeutic concentration and therapeutic duration (Table 40).

[0376] Combining the data of Examples 21 and 22, it can be seen that PEG400 can effectively promote the content of TTR in the vitreous body and fundus oculi samples, but it has comparatively poor therapeutic effects when used to treat DR rats or AMD mice. It is indicated that the increasing permeation of TTR does not mean the improvement of therapeutic effect. TTR, the appropriate permeation of TTR and appropriate excipient should mutual check and synergistically cooperate to form an organic whole, thus eventually achieving a better therapeutic effect.

[0377] In addition, it can be known from Table 25 and Table 27, even in normal SD rats, the retinal leakage phenomenon is more severe in adult SD rats than that in juvenile SD rats. In addition, other causes such as experimental operations and individual difference, also can lead to retinal leakage and increased number of neovascularization, which indicates that even normal control eyes without modeling have a certain degree of retinal leakage and increased neovascularization. By using TTR combined with chondroitin sulfate A sodium salt, it can not only treat DR, AMD and ROP, but also have the effect of nourishment, which has certain alleviation effect on retinal leakage and increased neovascularization caused by other reasons in an eye itself.

TABLE-US-00043 TABLE 40 Human TTR entering the vitreous body and fundus oculi through the corneal barrier of C57BL/6 mice and SD rats (1) The human TTR content in vitreous body and fundus oculi 3 h after eye dropping using human recombinant TTR (in saline solution). Concentration of TTR drops (mon) 5 10 15 20 25 30 Concentration of human TTR in vitreous 0.84 ± 1.81 ± 1.75 ± 1.83 ± 1.88 ± 1.87 ± body and fundus oculi samples of C57BL/6 0.07 0.19 0.20 0.21 0.17 0.20 mice (μmol/L) Concentration of human TTR in vitreous 1.14 ± 2.43 ± 2.37 ± 2.42 ± 2.45 ± 2.44 ± body and fundus oculi samples of SD rats 0.11 0.21 0.30 0.36 0.42 0.36 (μmol/L) (2) The human TTR content in vitreous body and fundus oculi 3 h after eye dropping using human recombinant TTR (10 μmol/L) (in saline solution added with PEG400 of different concentration). Concentration of PEG400 (mg/mL) 0 2 4 6 8 Concentration of human TTR in vitreous 1.81 ± 2.29 ± 2.83 ± 3.02 ± 3.00 ± 0.09 body and fundus oculi samples of C57BL/6 0.19 0.18 0.21 0.15 mice (μmol/L) Concentration of human TTR in vitreous 2.43 ± 2.99 ± 3.33 ± 3.51 ± 3.47 ± 0.36 body and fundus oculi samples of SD rats 0.21 0.21 0.29 0.32 (μmol/L) (3) The peak of the human TTR content in vitreous body and fundus oculi was reached 3 h after eye dropping using human recombinant TTR (10 μmol/L) (in saline solution added with 6 mg/mL PEG400), and the human TTR content thereof at different times thereafter are shown as below Sampling time after eye dropping (h) 3 18 36 60 72 Concentration of human TTR in vitreous 3.02 ± 2.61 ± 2.01 ± 1.50 ± 1.00 ± 0.09 body and fundus oculi samples of C57BL/6 0.15 0.20 0.18 0.12 mice (μmol/L) Concentration of human TTR in vitreous 3.51 ± 3.04 ± 2.13 ± 1.73 ± 1.14 ± 0.10 body and fundus oculi samples of SD rats 0.32 0.21 0.20 0.11 (μmol/L)

Comparative Example 7

[0378] The detailed operation manner is the same with Example 26 except the difference that, each ligand molecule not binding to TTR was used to conduct the eye dropping test on C57BL/6 mice (8 week old) and SD rats (8 week old) according to the operation steps which are the same with Example 26. The results show that each ligand molecule could not enter independently the vitreous body and fundus oculi of C57BL/6 mice and SD rats (Table 41).

TABLE-US-00044 TABLE 41 Each ligand molecule entering the vitreous body and fundus oculi through the corneal barrier of C57BL/6 mice and SD rats The content of each ligand in the vitreous body and fundus oculi at different times after eye dropping using saline solution added with 2% hyaluronic acid, and 1 mmol/L diclofenac sodium/1 mmol/L vitamin A + 5% Tween 80/1 mmol/L luteoloside. Sampling time after eye dropping (h) 3 18 36 60 72 Concentration of diclofenac sodium — — — — — in vitreous body and fundus oculi samples of C57BL/6 mice (μmol/L) Concentration of vitamin A in — — — — — vitreous body and fundus oculi samples of C57BL/6 mice (μmol/L) Concentration of luteoloside in — — — — — vitreous body and fundus oculi samples of C57BL/6 mice (μmol/L) Concentration of diclofenac sodium — — — — — in vitreous body and fundus oculi samples of SD rats μmol/L) Concentration of vitamin A in — — — — — vitreous body and fundus oculi samples of SD rats (μmol/L) Concentration of luteoloside in — — — — — vitreous body and fundus oculi samples of SD rats (μmol/L)

Comparative Example 8

[0379] The detailed operation manner is the same with Example 27, and the results of eye dropping in the right eye are shown in Table 42. The results show that each ligand molecule could not improve the DR pathological condition of SD rats induced by STZ with eye dropping independently (Table 42).

TABLE-US-00045 TABLE 42 DR pathological condition of SD rats induced by STZ treated with each ligand independently The DR pathological condition of SD rats induced by STZ after 3 months of eye dropping treatment using saline solution added with 2% hyaluronic acid, and 10 μmol/L diclofenac sodium/5 μmol/L vitamin A + 5% Tween 80/5 μmol/L luteoloside normal no diclo- control dropping fenac vitamin luteo- eye control sodium A loside Concentration of eye — 0 10 5 5 dropping (μmol/L) Retinal leakage area (%) 4.9 ± 29.4 ± 30.4 ± 28.9 ± 27.6 ± 0.5 3.1 3.1 3.0 4.1 No. of retinal 11 ± 2 77 ± 6 80 ± 9 76 ± 8 79 ± 9 neovascularization (10 visual fields)

Comparative Example 9

[0380] The detailed operation manner is the same with Example 28, and the results of eye dropping in the right eye are shown in Table 43. The results show that each ligand molecule could not improve the AMD pathological condition of C57BL/6 mice induced by laser retinal photocoagulation with eye dropping independently (Table 43).

TABLE-US-00046 TABLE 43 AMD pathological condition of C57BL/6 mice induced by laser retinal photocoagulation treated with each ligand independently The AMD pathological condition of C57BL/6 mice induced by laser retinal photocoagulation after 2 weeks of eye dropping treatment using saline solution added with 2% hyaluronic acid, and 10 μmol/L diclofenac sodium/5 μmol/L vitamin A + 5% Tween 80/5 μmol/L luteoloside normal no control dropping diclofenac vitamin luteo- eye control sodium A loside Concentration — 0 10 5 5 of eye dropping (μmol/L) Retinal leakage 3.9 ± 0.4 24.7 ± 2.2 25.2 ± 3.0 26.7 ± 3.4 21.3 ± 2.2 area (%) No. of retinal  11 ± 3     80 ± 7     85 ± 6     79 ± 10    82 ± 8   neovas- cularization (10 visual fields)

Comparative Example 10

[0381] The detailed operation manner is the same with Example 29, and the results of eye dropping in the right eye are shown in Table 44. The results show that each ligand molecule could not improve the ROP pathological condition of SD suckling rats induced by hyperbaric oxygen chamber with eye dropping independently (Table 44).

TABLE-US-00047 TABLE 44 ROP pathological condition of SD suckling rats induced by hyperbaric oxygen chamber treated with each ligand independently The ROP pathological condition of SD suckling rats induced by hyperbaric oxygen chamber after 5 days of eye dropping treatment using saline solution added with 2% hyaluronic acid, and 10 μmol/L diclofenac sodium/5 μmol/L vitamin A + 5% Tween 80/5 μmol/L luteoloside normal no control dropping diclofenac luteo- eye control sodium vitamin A loside Concentration — 0 10 5 5 of eye dropping (μmol/L) Retinal 3.3 ± 0.3 44.7 ± 5.1 48.5 ± 5.8 46.9 ± 4.9 51.2 ± 6.8 leakage area (%)

Comparative Example 11

[0382] Sulfamethoxazole has a broad antibacterial spectrum and strong antibacterial effect. It can block the growth of bacteria, particularly effective against Staphylococcus and Escherichia coli; it is suitable for respiratory, urinary and intestinal infections; it is mainly used to treat avian cholera, etc. It can be used to prepare an eye drops such as compound sulfamethoxazole sodium eye drops (this product is a compound preparation, each 10 mL contains 400 mg sulfamethoxazole sodium, 200 mg aminocaproic acid, 10 mg dipotassium glycyrrhizinate, 2 mg chlorpheniramine maleate. It is mainly used for bacterial conjunctivitis, blepharitis (stye) and bacterial blepharitis caused by sensitive bacteria.

[0383] Through molecular simulation with Discovery studio software, it is found that sulfamethoxazole can bind stably to a TTR polymer. In FIG. 17A, the protein structure is TTR dimer, sulfamethoxazole ligand molecule is represented by arrows, and one molecule of TTR dimer can bind to one molecule of sulfamethoxazole. FIG. 17B shows the interaction between sulfamethoxazole and amino acid residues of TTR.

[0384] In 10 μmol/L TTR solution (1000 μL), 100 μmol/L of sulfamethoxazole solution was dropped at the velocity of 1 μL/min according to the steps described in Example 25, and the affinity binding equilibrium dissociation constant K.sub.d was calculated with built-in software as 7.03×10.sup.−8 mol/L.

[0385] According to the steps described in Example 26, TTR/sulfamethoxazole sodium salts was used to treat rats and mice with eye dropping, and after 3-72 h, the cornea of rats and mice was damaged, i.e., sulfamethoxazole sodium salts used with TTR would burn the cornea and is not biologically safe. It can be seen that after screening for ligands having biological activity, having anti-inflammatory effects and having the ability to bind to TTR by molecular simulation with Discovery studio software, not all of the ligands can improve the therapeutic effect when verified by experiments.

[0386] Although the specific embodiments of the present disclosure are described above, those skilled in the art should understand that these are only examples, and various changes or modifications can be made to these embodiments without departing from the principle and essence of the present disclosure. Therefore, the protection scope of the present disclosure is defined by the appended claims.