CN-121971640-A - Preparation method of rebamipide-polypeptide conjugate and application of rebamipide-polypeptide conjugate in dry eye treatment

Abstract

The invention provides a preparation method of a rebamipide-polypeptide conjugate and application thereof in dry eye treatment, and belongs to the field of pharmaceutical chemistry. The rebamipide-polypeptide conjugate synthesized by the invention consists of three modules of a hydrophobic drug rebamipide, a beta-sheet self-assembly motif and a hydrophilic fragment. The drug is linked to the polypeptide by a cleavable disulfide bond. The resulting rebamipide-polypeptide conjugate can self-assemble in water to form a nanostructure, incorporating hydrophobic drugs therein. Under the action of ocular surface reduced glutathione, disulfide bonds connected with the medicine are broken, so that the medicine is released. The strategy utilizes the drug as a molecular building block for constructing a delivery carrier of the drug, obviously improves the solubility of the hydrophobic drug, prolongs the pre-cornea residence time of the drug, and realizes better treatment effect. The method is simple and convenient to synthesize, has strong repeatability, provides a foundation for application in the fields of drug delivery, biomedical engineering and the like, and has good application prospect.

Inventors

• SU HAO
• Xia Daowei
• ZHOU DONGDONG
• XIE JING

Assignees

• 四川大学

Dates

Publication Date

20260505

Application Date

20260203

Claims (10)

1. 1. The rebamipide-polypeptide conjugate, stereoisomer or pharmaceutically acceptable salt thereof is characterized by comprising rebamipide, self-assembled short peptide and cleavable linker, wherein the structure of the rebamipide-polypeptide conjugate is shown as formula I: I is a kind of Wherein R is selected from amino acid residues of glycine, valine, isoleucine, tyrosine, lysine, aspartic acid, and glutamine.
2. 2. The rebamipide-polypeptide conjugate, stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, wherein the structure of the rebamipide-polypeptide conjugate is as shown in formula II: II (II) Wherein R is selected from amino acid residues of glycine, valine, isoleucine, tyrosine, lysine, aspartic acid, and glutamine.
3. 3. The rebamipide-polypeptide conjugate, stereoisomer thereof or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the structure of the rebamipide-polypeptide conjugate is selected from one of the following structures: 。
4. 4. a method of preparing the rebamipide-polypeptide conjugate, stereoisomer or pharmaceutically acceptable salt thereof according to any one of claims 1-3, comprising the steps of: (1) Preparation of self-assembled short peptides: ; (2) Reacting self-assembled short peptide with rebamipide intermediate RBM-SS-Pyr to obtain rebamipide-polypeptide conjugate, wherein the RBM-SS-Pyr has the structure of 。
5. 5. The method according to claim 4, wherein in the step (1), the method for producing a self-assembled short peptide comprises the steps of swelling RINK AMIDE a resin in an organic solvent, deprotecting the protecting group using an alkali-containing deprotecting solution, adding an activated amino acid derivative for reaction, deprotecting the protecting group of the amino acid derivative using an alkali-containing deprotecting solution, repeating the above amide condensation process and deprotection process, attaching the amino acid derivatives to the resin one by one in the order of the designed polypeptide sequence, then, cleaving the polypeptide from the resin using an acid-containing eluent, precipitating the polypeptide with a poor solvent, centrifuging and discarding the supernatant, and purifying the precipitate by preparative high performance liquid chromatography after drying to obtain a self-assembled short peptide.
6. 6. The method according to claim 4, wherein in the step (2), the ratio of the amounts of the self-assembled short peptide and the RBM-SS-Pyr is 1:1.2-3, the solvent of the reaction is an organic solvent, the temperature of the reaction is 10-40 ℃ and the time is 4-24 hours; Preferably, the ratio of the self-assembled short peptide to the RBM-SS-Pyr is 1:1.5, the solvent of the reaction is dimethyl sulfoxide, the temperature of the reaction is 10-30 ℃, and the time is 6-24 hours.
7. 7. A method according to claim 3, wherein the preparation of RBM-SS-Pyr comprises the steps of: (a) Reacting 2-mercaptoethanol with 2,2' -dithiodipyridine to obtain Pyr-SS-OH; (b) Reacting rebamipide, pyr-SS-OH, an activator and a catalyst to obtain RBM-SS-Pyr.
8. 8. Use of a rebamipide-polypeptide conjugate according to any one of claims 1-3, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for alleviating and/or treating dry eye.
9. 9. A pharmaceutical composition, which is characterized in that the pharmaceutical composition is a pharmaceutical preparation prepared by taking the rebamipide-polypeptide conjugate, the stereoisomer or the pharmaceutically acceptable salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials according to any one of claims 1-3.
10. 10. The pharmaceutical composition of claim 9, wherein the pharmaceutical formulation is an ophthalmic drop formulation.

Description

Preparation method of rebamipide-polypeptide conjugate and application of rebamipide-polypeptide conjugate in dry eye treatment Technical Field The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a preparation method of a rebamipide-polypeptide conjugate and application of the rebamipide-polypeptide conjugate in dry eye treatment. Background Dry Eye (DED) is one of the common ocular diseases affecting about 30% of the population worldwide, bringing a tremendous economic burden to society. The main features of DED are loss of tear film homeostasis and ocular surface inflammation, and DED patients often exhibit foreign body sensation and blurred vision. The pathogenesis of DED is complex, including intrinsic and extrinsic factors, such as tear film instability and hyperopia, ocular surface inflammation and damage, and neural paresthesia. Rebamipide (RBM) is a quinolone derivative having mucin-promoting activity. In Japan, its ophthalmic suspension preparation (Mucosta ophthalmic suspension UD 2%, manufactured by Katsukamu pharmaceutical Co., ltd.) has been batched for the treatment of dry eye. Studies have shown that rebamipide can increase the number of corneal epithelial cells and conjunctival goblet cells of the eye, up-regulate the expression of mucin genes, promote the secretion of mucin, thereby protecting or stabilizing cornea and conjunctiva, and in addition, has an improving effect on corneal epithelial and conjunctival epithelial lesions, and thus, can improve or treat dry eye. However, due to poor solubility of the drug rebamipide, the ophthalmic suspension preparation of the drug is white suspension, particle aggregation and sedimentation can occur during long-term storage, and short-term fog vision can be caused after eye dropping, so that the vision of a patient is affected. In addition, due to blinking and tear clearance, the drug stays in the ocular surface for a short period of time, and the barrier of the anterior segment of the eye delays passive absorption of the drug, thereby reducing the ocular bioavailability (< 5%) of the drug. Long-term and high frequency dosing is often required, resulting in a substantial reduction in patient compliance. Therefore, the design of novel conjugates with good water solubility, ocular surface retention and controlled release behavior remains a technical problem to be further solved. Disclosure of Invention In order to overcome the problems in the prior art, the invention aims to provide a preparation method of a rebamipide-polypeptide conjugate and application of the rebamipide-polypeptide conjugate in dry eye treatment. The invention adopts the following technical scheme: The invention provides a rebamipide-polypeptide conjugate, a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the rebamipide-polypeptide conjugate consists of rebamipide, self-assembled short peptide and a cleavable linker, and the structure of the rebamipide-polypeptide conjugate is shown as a formula I: I is a kind of Wherein R is selected from amino acid residues of glycine (G), valine (V), isoleucine (I), tyrosine (Y), lysine (K), aspartic acid (D) and glutamine (Q). Further, the structure of the rebamipide-polypeptide conjugate is shown in formula II: II (II) Wherein R is selected from amino acid residues of glycine, valine, isoleucine, tyrosine, lysine, aspartic acid, and glutamine. Further, the structure of the rebamipide-polypeptide conjugate is selected from one of the following structures: 。 The invention also provides a method for preparing the rebamipide-polypeptide conjugate, the stereoisomer or the pharmaceutically acceptable salt thereof, which comprises the following steps: (1) Preparation of self-assembled short peptides: ; (2) Reacting self-assembled short peptide with rebamipide intermediate RBM-SS-Pyr to obtain rebamipide-polypeptide conjugate, wherein the RBM-SS-Pyr has the structure of 。 Further, in the step (1), the method for preparing the self-assembled short peptide comprises the steps of swelling RINK AMIDE resin in an organic solvent, removing a protecting group by using an alkali-containing deprotection solution, adding an activated amino acid derivative for reaction, removing the protecting group of the amino acid derivative by using the alkali-containing deprotection solution, repeating the amide condensation process and the deprotection process, connecting the amino acid derivatives to the resin one by one according to the designed polypeptide sequence, then cutting the polypeptide from the resin by using an acid-containing eluent, precipitating the polypeptide by using a poor solvent, centrifuging and discarding the supernatant, and purifying the precipitate by using a preparative high-performance liquid chromatography after drying to obtain the self-assembled short peptide. Further, in the step (1), a self-assembled short peptide is synthesized by a 9-fluoroethylene methoxycarbonyl (Fmoc) solid