Ophthalmic preparation as well as preparation method and application thereof

An ophthalmic preparation and ocular technology, applied in the field of protein engineering, can solve problems such as the unsatisfactory effect of transthyretin, and achieve the effect of improving the treatment effect and alleviating retinal leakage

Pending Publication Date: 2022-05-31
易舟(上海)生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Aiming at the shortcomings in the prior art that the effect of transthyretin (TTR) alone in the treatment of eye diseases is still not ideal, the present invention provides an ophthalmic preparation and its preparation method and application

Method used

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  • Ophthalmic preparation as well as preparation method and application thereof
  • Ophthalmic preparation as well as preparation method and application thereof
  • Ophthalmic preparation as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1 Recombinant preparation of human transthyretin (TTR)

[0066] (1) Construction of the recombinant plasmid pETx-rhaPBAD-ttr: The pET-21a plasmid (purchased from the ATCC China Culture Collection Center) was transformed and reconstructed (the difference in sequence between the reconstituted plasmid and the original pET-21a plasmid was About 75%, the specific sequence is shown in SEQ ID NO: 4), and the rhaPBAD promoter (Rhamna Tang inducible type) is used to replace the T7 promoter, and the human TTR optimized nucleic acid sequence (shown in SEQ ID NO: 2) is connected at the same time , the amino acid sequence of TTR is shown in SEQ ID NO:1), and the overall nucleic acid sequence of the resulting plasmid is shown in SEQ ID NO:3. After sequencing verification (the sequencing company is Nanjing GenScript Biotechnology Co., Ltd.), the construction was successful.

[0067] (2) Expression and purification of recombinant human TTR: transform the pETx-rhaPBAD-ttr pl...

Embodiment 2

[0070] Example 2 Computer Simulation of the Binding Form of TTR and Each Ligand Molecule

[0071] Refer to the static co-crystal model (PDF: 3CFQ) of TTR dimer and diclofenac in the PDB database, such as Figure 4A shown. Figure 4AIn , the protein structure is a TTR dimer, and the diclofenac ligand molecule is indicated by an arrow, and one molecule of TTR dimer can bind two molecules of diclofenac. Figure 4B The interaction of diclofenac with TTR amino acid residues is shown in .

[0072] Molecular simulation by Discovery studio software found that vitamin A1 (retinol) can stably bind to the hydrophobic channel of TTR polymer. Figure 5A The protein structure shown in is a TTR dimer, the vitamin A1 ligand molecule is indicated by an arrow, and one molecule of TTR dimer can bind one molecule of vitamin A1. Figure 5B The interaction of vitamin A1 with TTR amino acid residues is shown in .

[0073] Molecular simulation by Discovery studio software found that vitamin A2 (3...

Embodiment 3

[0076] Example 3 Determination of Dynamic Specific Binding Parameters Between TTR and Each Potential Ligand Molecule

[0077] Use nano ITC (TA) to measure the affinity binding equilibrium dissociation constant K of TTR and each ligand molecule mentioned in Example 2 or its salt d , in 10 μmol / L TTR solution (1000 μL), dropwise add 100 μmol / L of the above-mentioned various ligand solutions at a rate of 1 μL / min, and use the built-in software to calculate the affinity binding equilibrium dissociation constant K d (Table 2).

[0078] Table 2 The affinity binding equilibrium dissociation constants of various ligand molecules and TTR

[0079]

[0080] As shown in Table 2, the equilibrium dissociation constants of affinity binding between various ligand molecules and TTR are close to 10 -8 mol / L, which is close to the ability of monoclonal antibodies to recognize a single epitope, indicating that the above-mentioned ligand molecules can specifically recognize and bind to TTR. ...

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Abstract

The invention provides an ophthalmic preparation as well as a preparation method and application thereof. The invention discloses an ophthalmic preparation for inhibiting ocular retinal leakage and / or reducing the number of retinal neovascularization. The ophthalmic preparation comprises vitamin A or pharmaceutically acceptable salt thereof and transthyretin, the transthyretin is a protein composed of amino acids as shown in SEQ ID NO: 1. The content of the transthyretin is 5 to 30 [mu] mol / L; the content of the vitamin A or the pharmaceutically acceptable salt thereof is 2-10 [mu] mol / L. The invention also provides a preparation method and application of the ophthalmic preparation. The transthyretin and the compound in the ophthalmic preparation can cooperate with each other, so that the treatment effect on eye diseases related to eye angiogenesis and / or eye retina leakage can be remarkably improved when the ophthalmic preparation is used for treating the eye diseases.

Description

technical field [0001] The invention belongs to the technical field of protein engineering, and specifically relates to an ophthalmic preparation containing transthyretin and a specific compound, a preparation method and application thereof. Background technique [0002] Diclofenac and its sodium salt: Diclofenac and its sodium salt are non-steroidal anti-inflammatory drugs, which have obvious analgesic, anti-inflammatory and antipyretic effects. Diclofenac sodium eye drops are used to treat uveitis, keratitis, scleritis, and inhibit the formation of corneal neovascularization; treat inflammatory reactions after intraocular surgery, laser lauteroplasty, or various eye injuries; inhibit cataracts Miotic reaction during surgery; pain relief and anti-inflammation after excimer laser keratectomy; treatment of allergic eye diseases such as vernal conjunctivitis and seasonal allergic conjunctivitis; prevention and treatment of cataract and intraocular lens postoperative inflammati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K9/00A61P27/02A61P9/10A61K31/196A61K31/07A61K31/352
CPCA61K38/1709A61K31/196A61K31/07A61K31/352A61P27/02A61P9/10A61K9/0048A61K2300/00
Inventor 辛瑜
Owner 易舟(上海)生物医药有限公司
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