Preparation method of rebamipide-polypeptide conjugate and application of rebamipide-polypeptide conjugate in xerophthalmia treatment

Abstract

The invention provides a preparation method of a rebamipide-polypeptide conjugate and application of the rebamipide-polypeptide conjugate in xerophthalmia treatment, and belongs to the field of medicinal chemistry. The rebamipide-polypeptide conjugate synthesized by the invention is composed of three modules, namely a hydrophobic drug rebamipide, a beta-sheet self-assembly motif and a hydrophilic fragment. The medicine is connected with the polypeptide through a breakable disulfide bond. The obtained rebamipide-polypeptide conjugate can be self-assembled in water to form a nano structure, and a hydrophobic drug is contained in the nano structure. Under the action of ocular surface reductive glutathione, the disulfide bond connected with the medicine is broken, so that the release of the medicine is realized. According to the strategy, the drug is used as a molecular building block for building a delivery carrier of the drug, the solubility of the hydrophobic drug is obviously improved, the precorneal retention time of the drug is prolonged, and a better treatment effect is achieved. The method is simple and convenient in synthesis and high in repeatability, provides a foundation for application in the fields of drug delivery, biomedical engineering and the like, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a method for preparing a rebamipide-peptide conjugate and its application in the treatment of dry eye syndrome. Background Technology

[0002] Dry eye syndrome (DED) is a common eye disease affecting approximately 30% of the global population, imposing a significant economic burden on society. The main characteristics of DED are loss of tear film homeostasis and ocular surface inflammation. Patients with DED often experience a foreign body sensation and blurred vision. The pathogenesis of DED is complex, involving both internal and external factors, such as tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities.

[0003] Rebamipide (RBM) is a quinolone derivative with mucin-promoting activity. In Japan, its ophthalmic suspension formulation (Mucosta Ophthalmic Suspension UD 2%, Otsuka Pharmaceutical Co., Ltd.) has been approved for the treatment of dry eye. Studies have shown that rebamipide can increase the number of corneal epithelial cells and conjunctival goblet cells, upregulate mucin gene expression, and promote mucin secretion, thereby protecting or stabilizing the cornea and conjunctiva. Furthermore, it has an ameliorative effect on corneal and conjunctival epithelial damage, thus improving or treating dry eye.

[0004] However, due to the poor solubility of rebamipide, its ophthalmic suspension is a white, cloudy liquid. Long-term storage may lead to particle aggregation and sedimentation, potentially causing temporary fogging after instillation and affecting the patient's vision. Furthermore, due to blinking and tear clearance, the drug remains on the ocular surface for a short time, and the barrier in the anterior segment of the eye delays passive absorption, thus reducing ocular bioavailability (<5%). This often necessitates long-term and frequent administration, resulting in significantly reduced patient compliance.

[0005] Therefore, designing novel conjugates that combine good water solubility, ocular surface retention capacity, and controllable release behavior remains a technical problem that needs further resolution. Summary of the Invention

[0006] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide a method for preparing rebamipide-peptide conjugates and their application in the treatment of dry eye syndrome.

[0007] The present invention adopts the following technical solution: This invention provides a rebamipide-peptide conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the rebamipide-peptide conjugate is composed of rebamipide, a self-assembled short peptide, and a cleavable linker; the structure of the rebamipide-peptide conjugate is shown in Formula I: Formula I R is selected from amino acid residues of glycine (G), valine (V), isoleucine (I), tyrosine (Y), lysine (K), aspartic acid (D), and glutamine (Q).

[0008] Furthermore, the structure of the rebamipide-peptide conjugate is shown in Formula Ⅱ: Formula I R is selected from amino acid residues of glycine, valine, isoleucine, tyrosine, lysine, aspartic acid, and glutamine.

[0009] Furthermore, the structure of the rebamipide-peptide conjugate is selected from one of the following structures: .

[0010] The present invention also provides a method for preparing the above-mentioned rebamipide-peptide conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof, the preparation method comprising the following steps: (1) Preparation of self-assembled short peptides: ; (2) The self-assembled short peptide and the rebamipide intermediate RBM-SS-Pyr were reacted to obtain the rebamipide-peptide conjugate; the structure of the RBM-SS-Pyr is as follows: .

[0011] Further, in step (1), the method for preparing self-assembled short peptides includes the following steps: swelling Rink Amide resin in an organic solvent and removing the protecting group using an alkaline deprotection solution; adding activated amino acid derivatives to react and removing the protecting group of the amino acid derivatives using an alkaline deprotection solution; repeating the above amide condensation process and deprotection process, and linking the amino acid derivatives one by one to the resin according to the designed polypeptide sequence; subsequently, using an acid-containing elution buffer to remove the polypeptide from the resin, precipitating the polypeptide with a poor solvent, centrifuging and discarding the supernatant; drying the precipitate and purifying it by preparative high-performance liquid chromatography to obtain the self-assembled short peptide.

[0012] Furthermore, in step (1), a self-assembled short peptide is synthesized using the 9-fluorene methoxycarbonyl (Fmoc) solid-phase peptide synthesis method.

[0013] Further, in step (2), the molar ratio of the self-assembled short peptide to RBM-SS-Pyr is 1:1.2-3; the solvent for the reaction is an organic solvent; the temperature of the reaction is 10~40℃, and the time is 4~24 hours; Preferably, the molar ratio of the self-assembled short peptide to RBM-SS-Pyr is 1:1.5; the solvent for the reaction is dimethyl sulfoxide; the reaction temperature is 10~30℃, and the reaction time is 6~24 hours.

[0014] Furthermore, the preparation of the RBM-SS-Pyr includes the following steps: (a) Reaction of 2-mercaptoethanol and 2,2'-dithiopyridine yields Pyr-SS-OH; (b) Rebamipide, Pyr-SS-OH, activator and catalyst are reacted to obtain RBM-SS-Pyr.

[0015] Further, in step (a), the molar ratio of 2-mercaptoethanol to 2,2'-dithiopyridine is 1:1 to 3; the solvent for the reaction is an alcohol solvent; the reaction temperature is 10 to 40°C, and the reaction time is 2 to 20 hours; In step (b), the molar ratio of rebamipide, Pyr-SS-OH, activator and catalyst is 1.1~1.5:1:2~5:4~8; the solvent for the reaction is an organic solvent; the reaction temperature is 10~40℃ and the time is 4~24 hours. Preferably, in step (a), the molar ratio of 2-mercaptoethanol to 2,2'-dithiopyridine is 1:1.5~2; the solvent for the reaction is methanol; the reaction temperature is 15~35℃, and the reaction time is 3~10 hours; In step (b), the molar ratio of rebamipide, Pyr-SS-OH, activator and catalyst is 1.1:1:2.5:5; the solvent for the reaction is dichloromethane; the reaction temperature is 15~35℃ and the time is 4~15 hours.

[0016] The present invention also provides the use of the above-mentioned rebamipide-peptide conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of medicaments for relieving and / or treating dry eye syndrome.

[0017] The present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition is a pharmaceutical preparation prepared by adding pharmaceutically acceptable excipients to the above-mentioned rebamipide-peptide conjugate, its stereoisomer or its pharmaceutically acceptable salt as the active ingredient.

[0018] Furthermore, the pharmaceutical preparation is an ophthalmic drop preparation.

[0019] Preferably, the dry eye model is established by instilling benzalkonium chloride (BAC) into the ocular surface of Balb / c mice.

[0020] Preferably, the polypeptide drug conjugate solution is administered by infusion 4 times a day for 7 days.

[0021] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention, through rational molecular design, successfully constructs a novel class of rebamipide-peptide conjugates by covalently coupling rebamipide with a peptide module designed with a specific sequence via a breakable linker. The hydrophilic peptide fragment in this conjugate significantly improves the water solubility of rebamipide, potentially overcoming the physical stability defects and visual interference problems of existing suspension formulations. Simultaneously, the peptide sequence contains a β-sheet self-assembly motif, enabling the formation of ordered assemblies in the aqueous phase, thereby prolonging the drug's residence time on the ocular surface and reducing the frequency of administration. More importantly, the drug and peptide are linked by an environmentally sensitive disulfide bond, allowing for controlled drug release under microenvironmental stimuli such as ocular surface inflammation, improving local drug utilization. The synthetic method of the conjugate described in this invention combines solid-phase peptide synthesis with liquid-phase chemical coupling strategies, offering a clear and reproducible route, providing a new material basis and technical approach for developing highly effective and patient-friendly dry eye treatments.

[0022] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0023] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the synthesis scheme for rebamipide-peptide conjugates.

[0025] Figure 2 (A) HPLC and (B) LC-MS spectra of 2-(2-pyridyldithio)ethanol (Pyr-SS-OH).

[0026] Figure 3 The HPLC and LC-MS spectra of the intermediate RBM-SS-Pyr for riboplastin are shown in (A) and (B) respectively.

[0027] Figure 4 The HPLC and LC-MS spectra of rebamipide-peptide conjugate RBM-Tau-KK are shown in (A) and (B) respectively.

[0028] Figure 5The HPLC and LC-MS spectra of rebamipide-peptide conjugate RBM-Tau-KE are shown in (A) and (B) respectively.

[0029] Figure 6 The HPLC and LC-MS spectra of the rebamipide-peptide conjugate RBM-Tau-EE are shown in (A) and (B) respectively.

[0030] Figure 7 The HPLC and LC-MS spectra of rebamipide-peptide conjugate RBM-Tau-EK are shown in (A) and (B) respectively.

[0031] Figure 8 This is a transmission electron microscope image of the rebamipide-peptide conjugate.

[0032] Figure 9 The image shows a circular dichroism chromatogram of the rebamipide-peptide conjugate.

[0033] Figure 10 This is a graph showing the zeta potential results for rebamipide-peptide conjugates.

[0034] Figure 11 The image shows the CMC test results for rebamipide-peptide conjugate.

[0035] Figure 12 This is a diagram showing the drug release of rebamipide-peptide conjugate.

[0036] Figure 13 This is a diagram showing the results of corneal fluorescence staining.

[0037] Figure 14 This is a graph showing the results of a tear secretion test. Detailed Implementation

[0038] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0039] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0040] In this invention, "room temperature" means 25±5℃.

[0041] In this invention, "overnight" means a reaction time of 12 ± 5 hours.

[0042] Example 1: Preparation of rebamipide-peptide conjugate In this embodiment, rebamipide-peptide conjugates were prepared by the following method ( Figure 1 Specifically, it includes the following steps: (1) Synthesis of peptide sequences using Fmoc solid-phase synthesis: Synthesized on Rink Amide AM resin. The simplified procedure is as follows: The resin was swelled with DCM, and the Fmoc groups were deprotected twice (15 minutes each time) with a 20% 4-methylpyridine DMF solution. Each amino acid was coupled manually by adding amino acid / HBTU / DIEA to DMF at a molar ratio of 4:3.92:10 (relative to the resin) and shaking for at least 2 hours. The deprotection and coupling processes were repeated to add other amino acids to the peptide chain. Each step was followed by rinsing with DMF before proceeding to the next step. Alkyl chains were coupled in the same manner as amino acid coupling. Subsequently, the peptide was cleaved from the resin by adding a TFA / H2O / TIS standard solution (95:2.5:2.5 ratio) and shaking for 2 hours. The collected TFA solution was dried and concentrated under nitrogen, the crude product was washed three times with cold diethyl ether precipitation, and dried in a vacuum oven at room temperature. Sample preparation was performed using a 1260 Infinity II RP-HPLC instrument at a flow rate of 20 mL / min and an elution time of 20 min, monitored at 220 nm. The HPLC mobile phase consisted of water and acetonitrile (ACN) containing 0.1% (v / v) trifluoroacetic acid (TFA). The purity of the final product was determined by analytical RP-HPLC at a flow rate of 1 mL / min, with a mobile phase gradient from 15% to 100% ACN (0.1% TFA) over 15 min, monitored at 220 nm. All samples achieved a purity exceeding 98%. Figure 1 The polypeptides with different sequences shown are (Ac-CGVQIVYKK, Ac-CGVQIVYKE, Ac-CGVQIVYEE, Ac-CGVQIVYEK).

[0043] (2) Synthesis of rebamipide intermediate: 2,2'-dithiopyridine (1.5 eq) and 2-mercaptoethanol (1 eq) were dissolved in 100 mL and 50 mL of methanol, respectively. 2-mercaptoethanol was added dropwise to 2,2'-dithiopyridine under stirring. The reaction was carried out at room temperature for approximately 4 hours. The product was purified by column chromatography (EA / PE), rotary evaporated, and dried to obtain 2-(2-pyridyldithio)ethanol. The HPLC and LC-MS spectra of 2-(2-pyridyldithio)ethanol are shown below. Figure 2 As shown. Rebamipide (1.1 eq), 2-(2-pyridyldithio)ethanol (1 eq), and DMAP (5 eq) were dissolved in DCM, and EDC (2.5 eq) was added and reacted overnight. The product was purified by column chromatography (MeOH / DCM), rotary evaporated, and dried to obtain the rebamipide intermediate RBM-buSS-Pyr. The HPLC and LC-MS spectra of the rebamipide intermediate RBM-SS-Pyr are shown below. Figure 3 As shown.

[0044] (3) Synthesis of rebamipide-peptide conjugate: The peptide synthesized in step (1) was dissolved in DMSO with RBM-buSS-Pyr and subjected to a thiol-disulfide bond exchange reaction under nitrogen atmosphere for about 2 hours. The reaction solution was diluted with water and ACN and purified by preparative RP-HPLC to obtain the desired product. Figure 1 The different products shown are rebamipide-peptide conjugates.

[0045] The HPLC and LC-MS spectra of rebamipide-peptide conjugates RBM-Tau-KK, RBM-Tau-KE, RBM-Tau-EE, and RBM-Tau-EK are shown below. Figures 4-7 As shown.

[0046] The following experimental examples demonstrate the beneficial effects of the present invention.

[0047] Experimental Example 1: Characterization of the self-assembly structure and verification of drug release from rebamipide-peptide conjugates In this experimental example, the self-assembly structure and drug release capability of the rebamipide-peptide conjugate were investigated using the following methods.

[0048] 1. Experimental Methods (1) Microstructure of rebamipide-peptide conjugates was observed using transmission electron microscopy (TEM): The microstructure was acquired using a Talos F200i TEM (Thermo Scientific, USA) at an accelerating voltage of 200 kV. The detailed TEM measurement procedure was as follows: 6 μL of the corresponding solution was added to a carbon film copper mesh (300 mesh), and after 30 seconds, excess solution was absorbed from the edge of the copper mesh using a lint-free paper. Subsequently, 6 μL of 20 mg / mL uranium acetate aqueous solution was added for negative staining, and after 30 seconds, excess solution was absorbed to obtain the TEM sample. Each sample was placed at room temperature for more than 4 hours before TEM imaging to ensure complete solvent evaporation.

[0049] (2) The secondary structure of the rebamipide-peptide conjugate was observed using circular dichroism (CD) spectroscopy: CD measurements were performed using a Chirascan CD spectrometer (Applied Photophysics Ltd, UK) in the wavelength range of 180 nm to 260 nm with a bandwidth of 1 nm, using a 0.1 mm optical path quartz UV-Vis absorption cell (Hellma Analytics, Germany). The final results were obtained by averaging three repeated measurements for each sample, after subtracting background spectra.

[0050] (3) The zeta potential of rebapate-peptide conjugate was measured by nanoparticle size and zeta potential analysis: The zeta potential was analyzed by a zeta sizer Nano ZS nanoparticle potential analyzer (Malvin Panaco, UK). The final result was obtained by averaging three repeated measurements for each sample.

[0051] (4) Critical micelle concentration (CMC) of rebapeptide-peptide conjugates was measured using the Nile Red fluorescence method: The CMC was determined by the Nile Red method. Nile Red exhibits strong fluorescence in hydrophobic environments but is quenched in aqueous environments. When the monomer concentration exceeds the CMC, Nile Red interacts with the hydrophobic portion and emits fluorescence. Here, fluorescence emission spectra were obtained using a FluoroMax-4 fluorescence spectrometer (Horiba, Japan). First, 10 μL of 1 mM Nile Red ether solution was added to a centrifuge tube, and the tube was left in the dark overnight to allow the ether to evaporate. Subsequently, 1200 μL of sample solutions of different concentrations diluted from the stock solution were added to the tube, and the tube was incubated overnight at room temperature. Three sets of parallel emission spectra were recorded for all samples in the wavelength range of 580 to 720 nm at an excitation wavelength of 550 nm. Concentration-intensity conversion curves were plotted by comparing the intensity ratio of 635 nm (the emission peak of the dye in a hydrophobic environment) to 660 nm (the emission peak under aqueous conditions).

[0052] (5) Drug release behavior study: The degradation of the conjugate in the presence or absence of glutathione was assessed using RP-HPLC. In short, a 400 μM conjugate solution was freshly prepared with deionized water before the experiment, assembled at room temperature, and diluted to 200 μM with sodium phosphate buffer (pH 7.4, 20 mM) with or without glutathione (GSH, 20 mM). The solution was incubated at 37 °C, and samples were taken at 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 hours. Samples were stored at -20 °C for analysis. The amount of residual conjugate was monitored by RP-HPLC under the following conditions: flow rate of 1 mL / min, from initial 15% acetonitrile to 100% acetonitrile at 15 min, held for 5 min, and monitored at 220 nm. Data are plotted as percentages of the original concentration.

[0053] 2. Experimental Results The results are as follows Figure 8-12 As shown. By Figure 8 It can be seen that RBM-Tau-KK can spontaneously assemble into supramolecular nanofibers several micrometers long with an average diameter of 7.18±1.23 nm in an aqueous environment, while RBM-Tau-KE and RBM-Tau-EK form shorter fibers with larger diameters (12.56±2.64 nm and 10.3±2.24 nm), respectively. RBM-Tau-EE, however, cannot form ordered assemblies. The corresponding CD data ( Figure 9 The results show that RBM-Tau-KK exhibits a strong negative peak near 215 nm, indicating that the molecules form a β-sheet structure through highly ordered intermolecular hydrogen bonds, while RBM-Tau-EE exhibits a negative peak near 197 nm, indicating that it mainly exists in the form of random coil.

[0054] Depend on Figure 10 Based on the CMC results, the CMC values ​​of RBM-Tau-KK, RBM-Tau-KE, and RBM-Tau-EK were 1.5 μM, 26.43 μM, and 8 μM, respectively, while the CMC of RBM-Tau-EE exceeded 1 mM and could not be accurately measured. This indicates that RBM-Tau-EE cannot form stable aggregates under the current conditions.

[0055] Figure 11 Zeta data show that KK and EK are strongly positive, with values ​​of 51.6 ± 0.872 mV and 42.4 ± 0.529 mV, respectively, while KE and EE are negative, with values ​​of -32.9 ± 1.967 mV and -37.2333 ± 1.193 mV, respectively.

[0056] Depend on Figure 12 The results showed that GSH-induced RBM-Tau-KK prodrugs could undergo slow and sustained lysis, with 69.53 ± 7.82% of the RBM-Tau-KK prodrugs having been degraded within 24 hours. This indicates that the RBM-Tau-KK prodrugs can slowly release bioactive RBMs in the GSH-reducing environment of the ocular surface. In contrast, without GSH, the concentration of RBM-Tau-KK decreased by only about 10% within 24 hours due to hydrolysis. These results clearly demonstrate the effectiveness and high specificity of the GSH-responsive drug release behavior of RBM-Tau-KK. This is advantageous for achieving slow release in GSH-rich ocular surface environments.

[0057] Experimental Example 2: In vivo therapeutic efficacy verification of rebamipide-peptide conjugate In this experimental case, the in vivo therapeutic effects of rebamipide-peptide conjugate were investigated using the following methods.

[0058] 1. Experimental Methods (1) Eight-week-old female Balb / c mice were used to induce dry eye syndrome by instilling 5 μL of 0.4% BAC into both eyes twice daily for 7 days. Subsequently, 0.2% BAC solution was instilled twice daily to maintain the damage during the next 7 days of treatment.

[0059] (2) After dissolving and assembling the rebamipide-peptide conjugate prepared in Example 1, it was dripped into the conjunctival sac of model mice and administered 4 times a day for 7 days. Three control groups were set up, namely PBS group, rebamipide group, and rebamipide-peptide conjugate group. The effective drug concentration of the conjugate and rebamipide group was 5mM.

[0060] 2. Experimental Methods The results are as follows Figure 13 , 14 As shown. By Figure 13 It can be seen that, compared with the PBS group, both the rebamipide-peptide conjugate group and the rebamipide group repaired corneal epithelial damage to some extent, but the conjugate group achieved complete healing of the corneal defect. Figure 14 It was found that the conjugate group also promoted the fastest recovery of tear secretion (Day 7: 4.41±1.16 mm / 60s), which was superior to the rebamipide group in both speed and significance.

[0061] As can be seen from the above embodiments and experimental examples, this invention prepares rebamipide-peptide conjugates based on rebamipide using a combination of liquid-phase and solid-phase methods. The resulting rebamipide-peptide conjugates can self-assemble in water to form nanostructures, incorporating hydrophobic drugs within them. Under the action of reduced glutathione on the ocular surface, the disulfide bonds connecting the drug break, thereby releasing the drug. This strategy utilizes the drug as a molecular building block for constructing its own delivery carrier, significantly improving the solubility of hydrophobic drugs, prolonging the pre-corneal residence time of the drug, and achieving better therapeutic effects. This method is simple to synthesize, highly reproducible, and provides a foundation for applications in drug delivery, biomedical engineering, and other fields, showing promising application prospects.

Claims

1. A rebamipide-peptide conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof, characterized in that, The rebamipide-peptide conjugate is composed of rebamipide, a self-assembled short peptide, and a cleavable linker; the structure of the rebamipide-peptide conjugate is shown in Formula I: Formula I R is selected from amino acid residues of glycine, valine, isoleucine, tyrosine, lysine, aspartic acid, and glutamine.

2. The rebamipide-peptide conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The structure of the rebamipide-peptide conjugate is shown in Formula ⅠI: Formula I R is selected from amino acid residues of glycine, valine, isoleucine, tyrosine, lysine, aspartic acid, and glutamine.

3. The rebamipide-peptide conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, The structure of the rebapeptide-peptide conjugate is selected from one of the following structures: 。 4. A method for preparing the rebamipide-peptide conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: (1) Preparation of self-assembled short peptides: ； (2) The self-assembled short peptide and the rebamipide intermediate RBM-SS-Pyr were reacted to obtain the rebamipide-peptide conjugate; the structure of the RBM-SS-Pyr is as follows: .

5. The method according to claim 4, characterized in that, In step (1), the method for preparing self-assembled short peptides includes the following steps: swelling Rink Amide resin in an organic solvent and removing the protecting group using an alkaline deprotection solution; adding activated amino acid derivatives and reacting, and removing the protecting group of the amino acid derivatives using an alkaline deprotection solution; repeating the above amide condensation and deprotection processes, and linking the amino acid derivatives one by one to the resin according to the designed polypeptide sequence; subsequently, using an acid-containing elution buffer to remove the polypeptide from the resin, 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 the self-assembled short peptide.

6. The method according to claim 4, characterized in that, In step (2), the molar ratio of the self-assembled short peptide to RBM-SS-Pyr is 1:1.2-3; the solvent for the reaction is an organic solvent; the reaction temperature is 10~40℃ and the time is 4~24 hours. Preferably, the molar ratio of the self-assembled short peptide to RBM-SS-Pyr is 1:1.5; the solvent for the reaction is dimethyl sulfoxide; the reaction temperature is 10~30℃, and the reaction time is 6~24 hours.

7. The method according to claim 3, characterized in that, The preparation of the RBM-SS-Pyr includes the following steps: (a) Reaction of 2-mercaptoethanol and 2,2'-dithiopyridine yields Pyr-SS-OH; (b) Rebamipide, Pyr-SS-OH, activator and catalyst are reacted to obtain RBM-SS-Pyr.

8. Use of the rebamipide-peptide conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 3 in the preparation of a medicament for relieving and / or treating dry eye syndrome.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition is a pharmaceutical preparation made by adding pharmaceutically acceptable excipients to the rebamipide-peptide conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof as the active ingredient according to any one of claims 1 to 3.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical preparation is an ophthalmic drop preparation.

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