CN111920940B - Eye preparation and preparation method and application thereof - Google Patents

Abstract

The invention provides an ophthalmic preparation, which comprises transthyretin and pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials are selected from one or more of carboxymethyl cellulose or salts thereof, chondroitin sulfate or salts thereof, and dextran. The invention also provides a preparation method and application of the ophthalmic preparation. The auxiliary materials contained in the ophthalmic preparation of the invention and the transthyretin are mutually contained and cooperatively matched to form an organic whole, so that the ophthalmic preparation can be used for cooperatively treating eye diseases related to eye angiogenesis and/or eye retinal leakage and the like, and finally obtains better treatment effect.

Description

Eye preparation and preparation method and application thereof

Technical Field

The invention belongs to the technical field of protein engineering, and particularly relates to an ophthalmic preparation containing transthyretin, a preparation method thereof and application thereof in treating eye diseases related to ocular angiogenesis and/or ocular retinal leakage and the like.

Background

Treatment of ocular diseases requiring effective delivery of drugs to the eye, particularly age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, retinopathy of prematurity, glaucoma-induced nerve changes, and the like; however, effective delivery of drugs to the eye is challenging due to the greater barrier presented to the eye. Accordingly, recently, ocular drug delivery systems have received a wide range of attention.

Depending on the ocular anatomy, ocular barriers include primarily the tear barrier, the corneal/conjunctival barrier, and the blood-ocular barrier; the multiple barriers can effectively block the invasion of exogenous chemical and biological molecules while protecting eyes, but also bring great obstruction to the transmission of drugs. Currently, the common administration modes for eyes mainly include conventional eye drops, subconjunctival injection, scleral administration, and intravitreal injection. Wherein, the contact time of the conventional eye drops and the ocular surface is short, and due to the structure and the characteristics of the conventional eye drops, some exogenous drugs and proteins are difficult to effectively enter eyes; subconjunctival, scleral, and intravitreal injections often cause some trauma. In recent years, there have been reports of designing functional small peptides capable of effectively breaking through various ocular barriers to reach eyes, such as patents of zheng ying, xu xin et al (a new class of small peptides inhibiting neovascularization and applications thereof, ZL2013100527142), patents of suli et al (a class of small peptides inhibiting neovascularization and applications thereof, ZL201310058978.9) and patents of yang xiao lu et al (a small molecule polypeptide preventing and/or treating inflammatory responses and applications thereof, 201210581759.4).

The researches have certain limitations, intraocular injection can bring certain trauma, and the delivery efficiency of the functional small peptide is low and the half-life period is short, so that a thought needs to be developed to find a safer and more stable delivery mode from a protein or peptide segment which has a transport function and can break through a physiological barrier and exists in a human body.

CN111437398A discloses a transthyretin-containing drop, which can directly use transthyretin with transport function and break physiological barrier; however, the therapeutic effect of the composition for treating eye diseases still needs to be improved.

Disclosure of Invention

Aiming at the defects of poor treatment effect and the like of a preparation for treating eye diseases in the prior art, the invention provides an ophthalmic preparation containing transthyretin (TTR), a preparation method and application thereof, and also provides application of carboxymethyl cellulose or a salt thereof, chondroitin sulfate or a salt thereof and/or dextran in preparation of the ophthalmic preparation containing the transthyretin. The ophthalmic preparation comprises materials such as carboxymethyl cellulose or salts thereof, such as sodium carboxymethyl cellulose, chondroitin sulfate or salts thereof, such as chondroitin sulfate A sodium salt, dextran, such as dextran 70 and the like, and the materials and the transthyretin are mutually contained and cooperatively matched to form an organic whole, so that the ophthalmic preparation can be used for cooperatively treating eye diseases related to eye angiogenesis and/or eye retinal leakage, such as diabetic retinopathy, senile macular degeneration and/or retinopathy of prematurity, and finally obtains better treatment effect.

Those skilled in the art know only that materials such as carboxymethyl cellulose or its salt such as sodium carboxymethyl cellulose, chondroitin sulfate or its salt such as chondroitin sulfate a sodium salt, dextran such as dextran 70, etc. have moisturizing and the like effects, and they can be generally used for preparing eye drops to effectively relieve dryness of eyes and the like. However, the present inventors have unexpectedly found that materials such as carboxymethyl cellulose or salts thereof, e.g., sodium carboxymethyl cellulose, chondroitin sulfate or salts thereof, e.g., chondroitin sulfate a sodium salt, dextran, e.g., dextran 70, and the like, can be used to bind and cooperate with transthyretin to form an organic whole, so as to achieve a synergistic treatment effect on ocular diseases such as diabetic retinopathy, age-related macular degeneration, and/or retinopathy of prematurity, which are associated with ocular angiogenesis and/or ocular retinal leakage, and finally achieve a better therapeutic effect.

In order to solve the above technical problems, the present invention provides, in a first aspect, an ophthalmic preparation comprising transthyretin and a pharmaceutically acceptable excipient selected from one or more of "carboxymethylcellulose or a salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystalline form thereof", "chondroitin sulfate or a salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystalline form thereof", and "dextran or a salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystalline form thereof".

In the present invention, the term "pharmaceutically acceptable" generally means that the excipient is generally non-toxic, safe, and suitable for use by the patient. The "patient" is preferably a mammal, more preferably a human.

Preferably, the carboxymethyl cellulose or the salt thereof, the solvate of the pharmaceutically acceptable salt thereof, or the crystal form thereof has a viscosity of 800-1200 CP.

Preferably, the chondroitin sulfate is chondroitin sulfate A.

Preferably, the dextran is dextran 70. The molecular weight of dextran 70 is generally in the range of 64000-76000.

In the present invention, the "salt" in said "or its salt" is usually a pharmaceutically acceptable salt, which usually means a salt prepared from the compound of the present invention with a relatively nontoxic, pharmaceutically acceptable acid or base. When compounds of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the prototype of such compounds with a sufficient amount of a pharmaceutically acceptable base, in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to: lithium salt, sodium salt, potassium salt, calcium salt, aluminum salt, magnesium salt, zinc salt, bismuth salt, ammonium salt, and diethanolamine salt. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a prototype of such a compound with a sufficient amount of a pharmaceutically acceptable acid in neat solution or in a suitable inert solvent. The pharmaceutically acceptable acids include inorganic acids including, but not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, and the like. The pharmaceutically acceptable acids include organic acids including, but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, saccharic acid, formic acid, ethanesulfonic acid, pamoic acid (i.e. 4, 4' -methylene-bis (3-hydroxy-2-naphthoic acid)), amino acids (e.g. glutamic acid, arginine), and the like. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they may be converted to base addition salts or acid addition salts. See in particular Berge et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science 66:1-19(1977), or, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P.Heinrich Stahl and Camile G.Wermuth, ed., Wiley-VCH, 2002). In a preferred embodiment, the "salt" of the "carboxymethyl cellulose or its salt" may be a sodium salt or a calcium salt, such as sodium carboxymethyl cellulose. In a preferred embodiment, the "salt" of the "chondroitin sulfate or salt thereof" can be a sodium salt or a calcium salt, such as chondroitin sulfate A sodium salt.

Preferably, in the ophthalmic preparation, the concentration of the carboxymethyl cellulose or the salt thereof, the solvate of the pharmaceutically acceptable salt thereof, or the crystal form thereof is 0-8mg/mL but not 0 (i.e. 0 < concentration ≦ 8mg/mL), preferably 2, 4, 6, or 8 mg/mL.

Preferably, the concentration of the chondroitin sulfate or the salt thereof, the solvate of the pharmaceutically acceptable salt thereof, or the crystal form thereof in the ophthalmic preparation is 0-40mg/mL but not 0 (i.e., 0 < concentration ≦ 40mg/mL), preferably 10, 20, 30, or 40 mg/mL.

Preferably, in the ophthalmic preparation, the concentration of the dextran or the salt thereof, the solvate of the pharmaceutically acceptable salt thereof, or the crystal form thereof is 0-0.8mg/mL but not 0 (i.e. 0 < concentration ≦ 0.8mg/mL), preferably 0.2, 0.4, 0.6 or 0.8 mg/mL.

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

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

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

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

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

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

It will be appreciated by those skilled in the art that the co-ordination and results of The Transthyretin (TTR) with the pharmaceutically acceptable excipients described in the present invention are equally applicable to the TTR derivatives mentioned in the prior art (e.g., CN111437398A, incorporated herein in its entirety) such as rat TTR with the amino acid sequence shown in SEQ ID NO:5, mouse TTR with the amino acid sequence shown in SEQ ID NO:6, or human TTR-CL with the amino acid sequence shown in SEQ ID NO:7, which is the protein product after C-TNPKE loss of mature TTR.

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

Preferably, the ophthalmic preparation may further comprise other pharmaceutically acceptable excipients conventionally used in the art, such as physiological saline and the like. In the present invention, the amount of the physiological saline may be used according to the standard defined in the art.

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

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

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

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

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

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

In a preferred embodiment, the ophthalmic formulation consists of 10 μmol/L transthyretin, 6mg/mL sodium carboxymethylcellulose, and physiological saline.

In a preferred embodiment, the ophthalmic formulation consists of 10 μmol/L transthyretin, 0.4mg/mL dextran 70, and saline.

In a preferred embodiment, the ophthalmic formulation consists of 10 μmol/L transthyretin, 20mg/mL chondroitin sulfate A sodium salt and physiological saline.

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

In order to solve the above technical problems, the third aspect of the present invention provides a method for preparing an ophthalmic preparation according to the first aspect of the present invention, wherein a pharmaceutically acceptable excipient according to the first aspect of the present invention is mixed with the transthyretin.

In order to solve the above technical problems, according to a fourth aspect of the present invention, there is provided a use of a pharmaceutically acceptable excipient in the preparation of an ophthalmic preparation, wherein the pharmaceutically acceptable excipient is selected from one or more of "carboxymethylcellulose or a (pharmaceutically acceptable) salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystal form thereof", "chondroitin sulfate or a (pharmaceutically acceptable) salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystal form thereof", and "dextran or a (pharmaceutically acceptable) salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystal form thereof".

Preferably, the carboxymethyl cellulose or the salt thereof, the solvate of the pharmaceutically acceptable salt thereof, or the crystal form thereof has a viscosity of 800-1200 CP.

Preferably, the chondroitin sulfate is chondroitin sulfate A.

Preferably, the dextran is dextran 70.

Preferably, the "salt" of the "carboxymethyl cellulose or a salt thereof" may be a sodium salt or a calcium salt, for example, sodium carboxymethyl cellulose.

Preferably, the "salt" of the "chondroitin sulfate or salt thereof" may be a sodium salt or a calcium salt, such as chondroitin sulfate a sodium salt.

Preferably, in the ophthalmic preparation, the concentration of the carboxymethyl cellulose or the salt thereof, the solvate of the pharmaceutically acceptable salt thereof, or the crystal form thereof is 0-8mg/mL but not 0 (i.e. 0 < concentration ≦ 8mg/mL), preferably 2, 4, 6, or 8 mg/mL.

Preferably, the concentration of the chondroitin sulfate or the salt thereof, the solvate of the pharmaceutically acceptable salt thereof, or the crystal form thereof in the ophthalmic preparation is 0-40mg/mL but not 0 (i.e., 0 < concentration ≦ 40mg/mL), preferably 10, 20, 30, or 40 mg/mL.

Preferably, in the ophthalmic preparation, the concentration of the dextran or the salt thereof, the solvate of the pharmaceutically acceptable salt thereof, or the crystal form thereof is 0-0.8mg/mL but not 0 (i.e. 0 < concentration ≦ 0.8mg/mL), preferably 0.2, 0.4, 0.6 or 0.8 mg/mL.

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

Preferably, the ophthalmic preparation may further comprise other pharmaceutically acceptable excipients conventionally used in the art, such as physiological saline and the like.

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

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

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

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

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

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

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

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

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

In the ophthalmic preparation of the present invention, The Transthyretin (TTR) may be present alone and be contained and cooperated with the pharmaceutically acceptable excipients of the present invention, but those skilled in the art should understand that other active ingredients for treating ocular diseases, or fusion proteins prepared from the active ingredients and the transthyretin (such as the fusion protein described in CN 111437398A) for preparing the ophthalmic preparation, should also fall within the protection scope of the present invention. Therefore, in a preferred embodiment of the present invention, the ophthalmic preparation may further comprise other effective components for treating eye diseases, such as lysozyme (GenBank accession number is AAL69327.1), egg albumin, etc.

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

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

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

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

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

In the present invention, the pharmaceutically acceptable auxiliary materials and the like are generally commercially available, for example, from the pharmaceutical group.

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

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

The positive progress effects of the invention are as follows: the ophthalmic preparation comprises materials such as carboxymethyl cellulose or salts thereof, such as sodium carboxymethyl cellulose, chondroitin sulfate or salts thereof, such as chondroitin sulfate A sodium salt, dextran, such as dextran 70 and the like, and the materials and the transthyretin are mutually contained and cooperatively matched to form an organic whole, so that the ophthalmic preparation can be used for cooperatively treating eye diseases related to eye angiogenesis and/or eye retinal leakage, such as diabetic retinopathy, senile macular degeneration and/or retinopathy of prematurity, and finally obtains better treatment effect.

Drawings

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

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

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

Detailed Description

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

Example 1 recombinant preparation of human transthyretin (TTR)

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

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

TABLE 1 recombinant expression of TTR of human origin

Example 2 use of sodium carboxymethylcellulose as an adjunct to promote TTR passage through the corneal barrier

(1) The human TTR prepared in example 1 was prepared at 5 to 30 μmol/L (containing physiological saline), and C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) were each subjected to eye drop, sacrificed after 3 hours, and the vitreous body and fundus oculi sample were used to extract protein, and the content of human TTR in the vitreous body and fundus oculi sample of C57BL/6 mice and SD rats was measured by ELISA using rabbit anti-His-tag antibody as a primary antibody and donkey anti-rabbit antibody as a secondary antibody.

(2) The content of the human TTR in the vitreous body and fundus sample of the C57BL/6 mouse and SD rat was measured by ELISA using a rabbit anti-His-tag antibody as a primary antibody and a donkey anti-rabbit antibody as a secondary antibody by dropping the C57BL/6 mouse (8 weeks old) and SD rat (8 weeks old) with 10. mu. mol/L (containing physiological saline and 0-8mg/mL sodium carboxymethylcellulose (purchased from Chinese medicine, viscosity: 800-1200CP)) of the human TTR prepared in example 1 to give 3-72h of death. The result shows that the content of the humanized TTR in the vitreous body and the fundus sample reaches the highest peak after 3 hours of eye dropping; in the eye drops, after the sodium carboxymethyl cellulose is added, the content of the human TTR in the vitreous body and the fundus sample is obviously improved (by more than 10%), and when the content of the sodium carboxymethyl cellulose is 6mg/mL, the content of the human TTR in the vitreous body and the fundus sample reaches the highest peak after 3 hours of eye drop; after eye dropping, the half-life of the human TTR content in the vitreous body and fundus sample of C57BL/6 mouse and SD rat is close to 72h, which shows that the human TTR can effectively exist in the vitreous body and fundus sample for 72h, and has enough treatment concentration and treatment time (Table 2).

TABLE 2 humanized TTR crosses the corneal barrier of C57BL/6 mice and SD rats into the vitreous and fundus

Example 3 use of dextran 70 as an adjunct to promote TTR passage through the corneal barrier

(1) The human TTR prepared in example 1 was prepared at 5 to 30 μmol/L (containing physiological saline), and C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) were each subjected to eye drop, sacrificed after 3 hours, and the vitreous body and fundus oculi sample were used to extract protein, and the content of human TTR in the vitreous body and fundus oculi sample of C57BL/6 mice and SD rats was measured by ELISA using rabbit anti-His-tag antibody as a primary antibody and donkey anti-rabbit antibody as a secondary antibody.

(2) The content of the human-derived TTR in the vitreous humor and fundus oculi samples of the C57 8932/6 mice and SD rats (8 weeks old) was measured by ELISA using a mixture of 10. mu. mol/L (containing physiological saline and 0-0.8mg/mL dextran 70 (molecular weight 64000-76000 purchased from national drugs)) prepared in example 1, by dropping the mixture into the eye of each of the C57BL/6 mice (8 weeks old) and SD rats (8 weeks old), and after 3-72 hours, the mixture was sacrificed, and the vitreous humor and fundus oculi samples were collected to extract the protein, and the content of the human-derived TTR in the vitreous humor and fundus oculi samples of the C57BL/6 mice and SD rats was measured by ELISA. The result shows that the content of the humanized TTR in the vitreous body and the fundus sample reaches the highest peak after 3 hours of eye dropping; in the eye drops, after the dextran 70 is added, the content of the human TTR in the vitreous body and the fundus sample is obviously improved (by more than 15%), and when the content of the dextran 70 is 0.4mg/mL, the content of the human TTR in the vitreous body and the fundus sample reaches the highest peak after 3 hours of eye drop; after eye dropping, the half-life of the human TTR content in the vitreous body and fundus sample of C57BL/6 mouse and SD rat is close to 60h, which shows that the human TTR can effectively exist in the vitreous body and fundus sample for 60h, and has enough treatment concentration and treatment time (Table 3).

TABLE 3 transfer of human TTR across the corneal barrier of C57BL/6 mice and SD rats into the vitreous and fundus

Example 4 use of chondroitin sulfate A sodium salt as an adjunct promotes the passage of TTR across the corneal barrier

(1) The human TTR prepared in example 1 was prepared at 5 to 30 μmol/L (containing physiological saline), and C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) were each subjected to eye drop, sacrificed after 3 hours, and the vitreous body and fundus oculi sample were used to extract protein, and the content of human TTR in the vitreous body and fundus oculi sample of C57BL/6 mice and SD rats was measured by ELISA using rabbit anti-His-tag antibody as a primary antibody and donkey anti-rabbit antibody as a secondary antibody.

(2) The human TTR prepared in example 1 was prepared at 10 μmol/L (containing physiological saline and 0 to 40mg/mL chondroitin sulfate a sodium salt (purchased from a national drug)), and C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) were each dropped with eye, sacrificed after 3 to 72 hours, and the vitreous body and fundus oculi sample were used to extract protein, and the content of human TTR in the vitreous body and fundus oculi sample of C57BL/6 mice and SD rats was measured by ELISA using rabbit anti-His-tag antibody as a primary antibody and donkey anti-rabbit antibody as a secondary antibody. The result shows that the content of the humanized TTR in the vitreous body and the fundus sample reaches the highest peak after 3 hours of eye dropping; in the eye drops, after the chondroitin sulfate A sodium salt is added, the content of the human TTR in the vitreous body and the fundus sample is obviously improved (by over 17%), and when the content of the chondroitin sulfate A sodium salt is 20mg/mL, the content of the human TTR in the vitreous body and the fundus sample reaches the highest peak after 3 hours of eye drop; after eye dropping, the half-life of the human TTR content in the vitreous body and fundus sample of C57BL/6 mouse and SD rat is close to 72h, which shows that the human TTR can effectively exist in the vitreous body and fundus sample for 72h, and has enough treatment concentration and treatment time (Table 4).

TABLE 4 introduction of human TTR into the vitreous and fundus oculi across the corneal barrier of C57BL/6 mice and SD rats

EXAMPLE 5 treatment of DR (diabetic retinopathy) SD rats with human TTR-vehicle eye drops

SD rats of 8 weeks old are 200-250g in weight, fasted for 12-18h, injected with 2% STZ (60mg/kg) in the abdominal cavity, collected by tail cutting after 48h and 72h, tested by blood glucose test paper is higher than 16.7mM, and successfully molded to obtain DR SD rats. The DR SD rats are divided into 6 groups, 1 group is DR SD rats without any treatment (5 rats), the other 5 groups are 5 rats per group, the left eye and the right eye are respectively dripped for 2 times a day and 30 mu L each time, wherein, the left eye is dripped by the human TTR prepared in the example 1, or the left eye and the right eye are dripped by the human TTR prepared in the example 1 and auxiliary materials, specifically, 10 mu mol/L human TTR (physiological saline solution), 10 mu mol/L human TTR (physiological saline solution +6mg/mL carboxymethylcellulose sodium), 10 mu mol/L human TTR (physiological saline solution +6mg/mL PEG400), 10 mu mol/L human TTR (physiological saline solution +0.4mg/mL dextran 70), 10 mu mol/L human TTR (physiological saline solution +20mg/mL chondroitin sulfate A sodium salt) are respectively dripped, the right eye is formed by dropping a normal saline solution or a normal saline solution added with an auxiliary material, and is used as a control, specifically formed by dropping a normal saline solution, a normal saline solution +6mg/mL sodium carboxymethyl cellulose, a normal saline solution +6mg/mL PEG400, a normal saline solution +0.4mg/mL dextran 70 and a normal saline solution +20mg/mL chondroitin sulfate A sodium salt respectively. In addition, another 1 group of normal SD rats served as controls (5). After all SD rats are continuously raised for 3 months, retinas are stripped respectively, Evans Blue staining is carried out to observe the retinal vessel leakage condition, and Trypsin enzymolysis is carried out to observe the neovascular density. The result shows that after the SD rat is bred for 3 months under the induction of STZ, the retinal vascular leakage and the number of new blood vessels are obviously improved compared with the normal control, the eyeball retinal leakage phenomenon of dripping the humanized TTR is obviously inhibited, the number of the retinal new blood vessels is obviously reduced, and the clinical pathological phenomenon of DR is relieved. Among them, the effect of adding 10. mu. mol/L of human TTR (physiological saline solution +20mg/mL chondroitin sulfate A sodium salt) dropwise daily was the best (Table 5). The treatment effect of dripping 10 mu mol/L of human TTR (physiological saline solution +6mg/mL of PEG400) is relatively poor.

TABLE 5 treatment of pathological conditions of SD rats DR induced by STZ with human TTR/human TTR-adjuvant

EXAMPLE 6 treatment of AMD (age-related macular degeneration) C57BL/6 mice with human TTR-adjuvant in eye drops

9 weeks old C57/BL6 mice were photocoagulated to the retina with krypton laser (647nm) at 360mW, 50 μm in diameter for 0.05s at 8 photocoagulation points per eye to induce choroidal neovascularization and progressive proliferation to the retina to obtain AMD C57BL/6 mice. AMD C57BL/6 mice were divided into 6 groups, 1 group was AMD C57BL/6 mice without any treatment (5 mice), and the other 5 groups were 5 mice/group, and left and right eyes were each added with 30. mu.L of solution 2 times a day, wherein left eye was added with TTR derived from human prepared in example 1, or with TTR derived from human prepared in example 1 added with adjuvants, specifically 10. mu. mol/L of TTR derived from human (physiological saline solution +6mg/mL carboxymethylcellulose sodium), 10. mu. mol/L of TTR derived from human (physiological saline solution +6mg/mL PEG400), 10. mu. mol/L of TTR derived from human (physiological saline solution +0.4mg/mL dextran 70), 10. mu. mol/L of TTR derived from human (physiological saline solution +20mg/mL chondroitin sulfate A sodium salt), respectively, the right eye is formed by dropping a normal saline solution or a normal saline solution added with an auxiliary material, and is used as a control, specifically formed by dropping a normal saline solution, a normal saline solution +6mg/mL sodium carboxymethyl cellulose, a normal saline solution +6mg/mL PEG400, a normal saline solution +0.4mg/mL dextran 70 and a normal saline solution +20mg/mL chondroitin sulfate A sodium salt respectively. In addition, 1 additional group of normal C57BL/6 mice served as controls (5). After 2 weeks of eye drop, the animals were sacrificed, the retinas were stripped and subjected to Evans Blue staining for retinal vessel leakage and Trypsin enzymatic digestion for neovascular density.

The results showed that more vadose areas appeared in the control group of AMD C57BL/6 mice, the number of new blood vessels increased significantly, and TTR eye drops reversed this trend (Table 6), and the treatment effect of TTR was better in the group to which sodium carboxymethylcellulose, dextran 70 and chondroitin sulfate A sodium salt were added.

TABLE 6 treatment of AMD pathological conditions in mice with human TTR/human TTR-adjuvant induced by laser retinal photocoagulation C57BL/6

EXAMPLE 7 treatment of ROP (retinopathy of prematurity) SD rats with human TTR-vehicle eye drops

One week of birth of the suckling mice (SD rats) were placed in a hyperbaric oxygen chamber and the normal control group was placed in a normal environment (normal control group, 5 mice). The rat was raised in a hyperbaric oxygen chamber for 5 days and then taken out to obtain ROP SD rat. All ROP SD rats were kept in normal environment for 5 days together with normal control group. In the 5 days, ROP SD rats are divided into 6 groups, 1 group is ROP SD mice without any treatment (5 mice), the other 5 groups are 5 mice per group, the left eye and the right eye are respectively dripped 1 time per day, 30 mu L each time, wherein the left eye is the eye drop of the human TTR prepared in the example 1, or the eye drop of the human TTR prepared in the example 1 and auxiliary materials, specifically 10 mu mol/L human TTR (physiological saline solution), 10 mu mol/L human TTR (physiological saline solution +6mg/mL carboxymethylcellulose sodium), 10 mu mol/L human TTR (physiological saline solution +6mg/mL PEG400), 10 mu mol/L human TTR (physiological saline solution +0.4mg/mL dextran 70), 10 mu mol/L human TTR (physiological saline solution +20mg/mL chondroitin sulfate A sodium salt) are respectively dripped, the right eye is formed by dropping a normal saline solution or a normal saline solution added with an auxiliary material, and is used as a control, specifically formed by dropping a normal saline solution, a normal saline solution +6mg/mL sodium carboxymethyl cellulose, a normal saline solution +6mg/mL PEG400, a normal saline solution +0.4mg/mL dextran 70 and a normal saline solution +20mg/mL chondroitin sulfate A sodium salt respectively. After 5 days of eye drop, the animals were sacrificed and the retinal vessels were observed for leakage by peeling off the retina and performing Evans Blue staining. After the malformed neovasculature in the ROP SD rat control group was engulfed with the retina, a large number of areas of leakage appeared, and the TTR eye drops reversed this trend (table 7), and the treatment effect of TTR was better in the group to which sodium carboxymethylcellulose, dextran 70 and sodium chondroitin sulfate a were added.

TABLE 7 pathological conditions of hyperbaric oxygen chamber induced SD rat suckling rat ROP treated by humanized TTR/humanized TTR-adjuvant

Comparative example 1

(1) The human TTR prepared in example 1 was prepared at 5 to 30 μmol/L (containing physiological saline), and C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) were each subjected to eye drop, sacrificed after 3 hours, and the vitreous body and fundus oculi sample were used to extract protein, and the content of human TTR in the vitreous body and fundus oculi sample of C57BL/6 mice and SD rats was measured by ELISA using rabbit anti-His-tag antibody as a primary antibody and donkey anti-rabbit antibody as a secondary antibody.

(2) The human TTR prepared in example 1 was prepared at 10. mu. mol/L (containing physiological saline and 0-8mg/mL PEG400 (purchased from national medicine, molecular weight: 360-440)), and C57BL/6 mice (8 weeks old) and SD rats (8 weeks old) were dropped into the eye, sacrificed after 3-72 hours, and the vitreous body and fundus oculi sample were used to extract protein, and the content of human TTR in the vitreous body and fundus oculi sample of C57BL/6 mice and SD rats was measured by ELISA using rabbit anti-His-tag antibody as a primary antibody and donkey anti-rabbit antibody as a secondary antibody. The result shows that the content of the humanized TTR in the vitreous body and the fundus sample reaches the highest peak after 3 hours of eye dropping; in the eye drops, after PEG400 is added, the content of human TTR in vitreous and fundus samples is improved by more than 20%, and when the content of PEG400 is 6mg/mL, the content of human TTR in vitreous and fundus samples reaches the highest peak after 3 hours of eye dropping; after eye dropping, the half-life of the human TTR content in the vitreous body and fundus sample of C57BL/6 mouse and SD rat is close to 60h, which shows that the human TTR can effectively exist in the vitreous body and fundus sample for 60h, and has enough treatment concentration and treatment time (Table 8).

Combining the results of examples 5 and 6, it is clear that although PEG400 is effective in promoting TTR levels in vitreous and fundus samples, it is relatively ineffective when used to treat mice with DR or AMD. This indicates that the increase of the amount of TTR penetration does not mean the increase of the therapeutic effect, and the appropriate amount of TTR, TTR penetration and appropriate excipients are mutually contained and cooperatively matched to form an organic whole, so that a better therapeutic effect can be finally obtained.

In addition, as can be seen from tables 5 and 7, even in normal SD rats, the retinal leakage of adult SD rats is more severe than that of young SD rats, and in addition, other reasons, such as experimental procedures, individual differences, etc., may cause the retinal leakage and the number of new blood vessels to increase, which indicates that there is a certain degree of retinal leakage and new blood vessels to increase even in the normal control eyes without modeling. After TTR and chondroitin sulfate A sodium salt are used in a matching way, DR, AMD and ROP can be treated, the eye patch can also play a nourishing role, and the eye patch has a certain relieving effect on retinal leakage and neovascularization increase caused by other reasons.

TABLE 8 transfer of human TTR across the corneal barrier of C57BL/6 mice and SD rats into the vitreous and fundus

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

SEQUENCE LISTING

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

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

<130> P20014218C

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 127

<212> PRT

<213> Homo sapiens

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 2

<211> 381

<212> DNA

<213> Artificial Sequence

<220>

<223> TTR mature fragment optimized nucleic acid sequence

<400> 2

ggtccgaccg gtaccggtga atctaaatgc ccgctgatgg ttaaagttct ggacgctgtt 60

cgtggttctc cggctatcaa cgttgctgtt cacgttttcc gtaaagctgc tgacgacacc 120

tgggaaccgt tcgcttctgg taaaacctct gaatctggtg aactgcacgg tctgaccacc 180

gaagaagaat tcgttgaagg tatctacaaa gttgaaatcg acaccaaatc ttactggaaa 240

gctctgggta tctctccgtt ccacgaacac gctgaagttg ttttcaccgc taacgactct 300

ggtccgcgtc gttacaccat cgctgctctg ctgtctccgt actcttactc taccaccgct 360

gttgttacca acccgaaaga a 381

<210> 3

<211> 4693

<212> DNA

<213> Artificial Sequence

<220>

<223> plasmid Whole nucleic acid sequence

<400> 3

aattcttaag aaggagatat acatatgaga ggatcgcatc atcatcatca tcatggatcc 60

ggtccgaccg gtaccggtga atctaaatgc ccgctgatgg ttaaagttct ggacgctgtt 120

cgtggttctc cggctatcaa cgttgctgtt cacgttttcc gtaaagctgc tgacgacacc 180

tgggaaccgt tcgcttctgg taaaacctct gaatctggtg aactgcacgg tctgaccacc 240

gaagaagaat tcgttgaagg tatctacaaa gttgaaatcg acaccaaatc ttactggaaa 300

gctctgggta tctctccgtt ccacgaacac gctgaagttg ttttcaccgc taacgactct 360

ggtccgcgtc gttacaccat cgctgctctg ctgtctccgt actcttactc taccaccgct 420

gttgttacca acccgaaaga atgaaagctt ctgttttggc ggatgagaga agattttcag 480

cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg 540

cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc 600

cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac 660

gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc 720

tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag 780

ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag aaggccatcc 840

tgacggatgg cctttttgcg tttctacaaa ctcttttgtt tatttttcta aatacattca 900

aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 960

aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 1020

cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 1080

ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 1140

cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 1200

ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 1260

gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 1320

gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 1380

acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 1440

cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 1500

acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 1560

ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 1620

ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 1680

gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 1740

atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 1800

ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 1860

attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 1920

ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 1980

aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 2040

aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 2100

ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 2160

tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 2220

ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 2280

cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 2340

agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 2400

gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 2460

ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 2520

tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 2580

tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 2640

cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 2700

tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 2760

gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 2820

atatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac 2880

tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga 2940

cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 3000

cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg 3060

gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc 3120

cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt 3180

aagggcggtt ttttcctgtt tggtcacttg atgcctccgt gtaaggggga atttctgttc 3240

atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat 3300

gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg 3360

gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt 3420

ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct 3480

gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct 3540

caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca 3600

ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg 3660

atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg 3720

gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc 3780

cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg 3840

cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga 3900

caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg 3960

cataaatcgc cgtgacgatc agcggtccag tgatcgaagt taggctggta agagccgcga 4020

gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca 4080

acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc 4140

ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa 4200

tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg 4260

cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc 4320

ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa 4380

agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga 4440

ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc cttatgcgac tcctgcatta 4500

ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat 4560

gcatcgatca ccacaattca gcaaattgtg aacatcatca cgttcatctt tccctggttg 4620

ccaatggccc attttcctgt cagtaacgag aaggtcgcga attcaggcgc tttttagact 4680

ggtcgtaatg aac 4693

<210> 4

<211> 4311

<212> DNA

<213> Artificial Sequence

<220>

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

<400> 4

aattcttaag aaggagatat acatatggga tcccatcatc atcatcatca ttgactgcag 60

ccaagcttct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa 120

cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct 180

gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc 240

catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg 300

ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc 360

gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc 420

ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt 480

tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 540

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 600

tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 660

aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 720

aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 780

tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc 840

aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 900

tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 960

ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 1020

taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 1080

agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 1140

caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 1200

tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 1260

gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 1320

cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 1380

caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 1440

ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 1500

aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 1560

gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1620

atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1680

tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 1740

gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 1800

actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 1860

gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 1920

agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 1980

ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 2040

aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 2100

cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2160

gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2220

cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 2280

cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 2340

gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt 2400

attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac 2460

aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg 2520

gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 2580

ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 2640

ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg 2700

tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga 2760

agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg 2820

gtcacttgat gcctccgtgt aagggggaat ttctgttcat gggggtaatg ataccgatga 2880

aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac 2940

gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg 3000

gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc 3060

ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt 3120

acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc 3180

agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac 3240

cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg 3300

acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata 3360

tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa 3420

ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc 3480

ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac 3540

aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag 3600

cggtccagtg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg 3660

atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc 3720

ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa 3780

gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg 3840

tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc 3900

aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca 3960

gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc 4020

gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg 4080

catcggtcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt 4140

gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc atcgatcacc acaattcagc 4200

aaattgtgaa catcatcacg ttcatctttc cctggttgcc aatggcccat tttcctgtca 4260

gtaacgagaa ggtcgcgaat tcaggcgctt tttagactgg tcgtaatgaa c 4311

<210> 5

<211> 127

<212> PRT

<213> Rattus norvegicus

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 6

<211> 127

<212> PRT

<213> Mus musculus

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 7

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> human TTR-CL

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val

115 120

Claims (16)

1. An ophthalmic preparation, comprising transthyretin and a pharmaceutically acceptable excipient selected from one of carboxymethylcellulose or a salt thereof, chondroitin sulfate or a salt thereof, and dextran;

the concentration of the carboxymethyl cellulose or the salt thereof is 6-8 mg/mL, the concentration of the chondroitin sulfate or the salt thereof is 20-40 mg/mL, the concentration of the dextran is 0.4-0.8 mg/mL, and the content of the transthyretin is 10-30 mu mol/L;

wherein, the chondroitin sulfate is chondroitin sulfate A, the dextran is dextran 70, the salt in the carboxymethyl cellulose or the salt thereof is sodium carboxymethyl cellulose, and the salt in the chondroitin sulfate or the salt thereof is chondroitin sulfate A sodium salt;

the transthyretin is shown as (a):

(a) 1, and a protein consisting of amino acids shown in SEQ ID NO.

2. The ophthalmic formulation of claim 1, wherein the carboxymethyl cellulose or a salt thereof has a viscosity of 800-1200 CP.

3. The ophthalmic formulation of claim 1, wherein the concentration of the carboxymethyl cellulose or its salt is 6 or 8 mg/mL;

the concentration of the chondroitin sulfate or the salt thereof is 20, 30 or 40 mg/mL;

the dextran concentration is 0.4, 0.6 or 0.8 mg/mL.

4. The ophthalmic formulation of claim 1, wherein the nucleotide sequence encoding the protein of (a) is set forth in SEQ ID NO. 2.

5. An ophthalmic formulation according to any one of claims 1 to 4, wherein the transthyretin is present in the ophthalmic formulation in an amount of 10, 15 or 20 μmol/L;

and/or, the ophthalmic preparation also contains physiological saline;

and/or, the ophthalmic formulation is a drop;

and/or, the ophthalmic preparation is an ophthalmic preparation for inhibiting ocular retinal leakage and/or reducing retinal neovascularization count;

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

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

7. The ophthalmic formulation of claim 5, wherein the drops are eye drops or ophthalmic liposomes;

and/or, the ophthalmic preparation is an ophthalmic preparation for treating diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity;

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

8. An ophthalmic formulation according to any one of claims 1 to 4, wherein the ophthalmic formulation is a spray or gel.

9. Use of an ophthalmic formulation according to any one of claims 1 to 8 for the preparation of a medicament for inhibiting ocular retinal leakage and/or reducing retinal neovascularisation.

10. The use according to claim 9, wherein the ophthalmic formulation is for the preparation of a medicament for the treatment of diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity.

11. A process for the preparation of an ophthalmic formulation according to any one of claims 1 to 8, characterized in that a pharmaceutically acceptable excipient according to any one of claims 1 to 8 is mixed with said transthyretin.

12. Use of a pharmaceutically acceptable excipient in the preparation of an ophthalmic formulation, wherein the ophthalmic formulation comprises transthyretin, and the pharmaceutically acceptable excipient is selected from one of carboxymethylcellulose or a salt thereof, chondroitin sulfate or a salt thereof, and dextran;

the concentration of the carboxymethyl cellulose or the salt thereof is 6-8 mg/mL, the concentration of the chondroitin sulfate or the salt thereof is 20-40 mg/mL, the concentration of the dextran is 0.4-0.8 mg/mL, and the content of the transthyretin is 10-30 mu mol/L;

the chondroitin sulfate is chondroitin sulfate A, the dextran is dextran 70, the salt in the carboxymethyl cellulose or the salt thereof is sodium carboxymethyl cellulose, and the salt in the chondroitin sulfate or the salt thereof is chondroitin sulfate A sodium salt;

the transthyretin is shown as (a):

(a) 1, and a protein consisting of amino acids shown in SEQ ID NO.

13. The use of claim 12, wherein the carboxymethyl cellulose or salt thereof has a viscosity of 800-;

and/or the concentration of the carboxymethyl cellulose or the salt thereof is 6 or 8 mg/mL;

and/or the concentration of the chondroitin sulfate or the salt thereof is 20, 30 or 40 mg/mL;

and/or the dextran concentration is 0.4, 0.6 or 0.8 mg/mL;

and/or the content of the transthyretin is 10, 15 or 20 mu mol/L;

and/or, the ophthalmic preparation also contains physiological saline;

and/or, the ophthalmic formulation is a drop;

and/or, the ophthalmic preparation is an ophthalmic preparation for inhibiting ocular retinal leakage and/or reducing retinal neovascularization count;

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

14. The use of claim 13, wherein the drops are eye drops or ophthalmic liposomes;

and/or, the ophthalmic preparation is an ophthalmic preparation for treating diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity;

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

15. The use of claim 12, wherein the ophthalmic formulation is a spray or gel.

16. The use of claim 12, wherein the nucleotide sequence encoding the protein of (a) is as set forth in SEQ ID NO 2.

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