A corneal injury repairing solution containing an active pentapeptide, and a preparation method and application thereof

Abstract

The application provides a corneal injury repair liquid containing an active pentapeptide and a preparation method and application thereof, and belongs to the technical field of biological medical materials. The active pentapeptide eye drop liquid comprises the following components: 10-100 mg / L of the active pentapeptide, 10-50 μg / L of recombinant human nerve growth factor, 5-25 μg / L of recombinant human keratinocyte growth factor-2, 1-10 mg / L of osmund acetate, 0.05-0.5 g / L of clitoria ternatea aqueous extract and 1.0-3.0 g / L of sodium hyaluronate; and the amino acid sequence of the active pentapeptide is REGRT. The corneal repair liquid provided by the application realizes rapid and high-quality repair of corneal injury through synergistic effect from six dimensions of 'promoting migration, promoting proliferation, promoting nerve repair, anti-inflammation, anti-oxidation and prolonging retention'. Not only can the corneal repair liquid prevent the occurrence of stromal edema and damage of epithelial barrier function, but also is crucial for long-term health maintenance of the recipient ocular surface after artificial cornea surgery.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a corneal damage repair solution containing active pentapeptides, its preparation method, and its application. Background Technology

[0002] The cornea, the transparent tissue at the very front of the eyeball, is directly exposed to the external environment and is susceptible to damage from physical friction, chemical burns, microbial infections, surgical trauma, and other factors. If corneal epithelial damage is delayed or fails to heal, it can develop into corneal ulcers or even perforation, severely affecting vision. Current clinical treatments for corneal injuries mainly include artificial tears, growth factor eye drops, and amniotic membrane transplantation. Existing eye drops often employ a single mechanism of intervention, such as using growth factors to promote epithelial repair or using nonsteroidal anti-inflammatory drugs (NSAIDs) to control inflammation. This makes it difficult to achieve multi-target synergistic treatment and simultaneously address: 1. rapid epithelial healing; 2. inhibition of pathological neovascularization and scarring; 3. relief of severe postoperative neuralgia; and 4. prevention of secondary bacterial / fungal infections. Therefore, there is an urgent need in this field for a multi-functional eye drop that combines functions such as promoting repair, analgesia, and anti-inflammation. Summary of the Invention

[0003] The purpose of this invention is to provide a corneal damage repair solution containing active pentapeptides, its preparation method, and its application.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a corneal injury repair solution containing an active pentapeptide, comprising: 10-100 mg / L of active pentapeptide, 10-50 μg / L of recombinant human nerve growth factor (NGF), 5-25 μg / L of recombinant human keratinocyte growth factor-2 (KGF-2), 1-10 mg / L of scutellarin, 0.05-0.5 g / L of butterfly pea flower water extract, and 1.0-3.0 g / L of sodium hyaluronate; The amino acid sequence of the active pentapeptide is: REGRT (SEQ ID NO.1).

[0005] Preferably, the preparation method of the butterfly pea flower water extract is as follows: take butterfly pea flower petals, add water at a material-to-liquid ratio of 1g:12~15mL, extract at 45~55℃ for 30~40min, filter, concentrate the filtrate under reduced pressure to a relative density of 1.05~1.10, freeze dry, and obtain butterfly pea flower extract.

[0006] Preferably, the corneal damage repair solution has a pH value of 6.5 to 7.5 and an osmotic pressure of 280 to 320 mOsm / L.

[0007] The present invention also provides a method for preparing a corneal damage repair solution containing active pentapeptides, specifically as follows: after preheating water for injection, sodium hyaluronate is added and swelled at room temperature for 8-12 hours. Then, scutellarin and butterfly pea flower water extract are added and stirred evenly. At 15-20°C, active pentapeptides, recombinant human nerve growth factor (NGF) and recombinant human keratinocyte growth factor-2 are added, pH and osmotic pressure are adjusted, and the solution is filtered for sterilization to obtain the corneal damage repair solution.

[0008] The present invention also provides the application of the repair solution in the preparation of products for corneal damage or postoperative repair and relief of corneal inflammation.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The active pentapeptide REGRT of this invention can significantly promote the migration of corneal epithelial cells and accelerate the closure of epithelial defects; NGF can promote corneal nerve repair and neurotrophic recovery, and KGF-2 can promote corneal epithelial cell proliferation and wound healing. These three components synergistically promote the structural reconstruction and functional recovery of corneal tissue from different aspects. Butterfly pea flower water extract not only has antioxidant activity but also promotes corneal epithelial cell migration. Together with scutellarin, it endows the repair solution with good anti-inflammatory and antioxidant activity, helping to reduce inflammatory reactions after corneal injury and improve the ocular surface microenvironment. Using sodium hyaluronate as a matrix can improve the ocular surface retention time and bioavailability of the active ingredients. The corneal repair solution provided by this invention works synergistically from six dimensions: promoting migration, promoting proliferation, promoting nerve repair, anti-inflammation, anti-oxidation, and prolonging retention, to achieve rapid and high-quality repair of corneal damage. It not only prevents stromal edema and damage to the epithelial barrier function but is also crucial for the long-term health maintenance of the recipient's ocular surface after artificial cornea surgery. Detailed Implementation

[0010] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0011] Example 1

[0012] Preparation method of active pentapeptide (REGRT): Weigh 1.0 g of Fmoc-Thr(t-Bu)-Wang resin (degree of substitution 0.4 mmol / g) and place it in a solid-phase synthesis tube. Add 10 mL of N,N-dimethylformamide (DMF) and bubble it under nitrogen at room temperature for 30 min. Add 10 mL of 20% piperidine / DMF solution to the synthesis tube in two portions, and elute and protect for 5 min and 15 min respectively. Filter and wash three times with DCM. Weigh 3 equivalents of Fmoc-Arg(Pbf)-OH, 3 equivalents of HBTU, and 3 equivalents of HOBt into a centrifuge tube, add 5 mL of DMF to dissolve them, add 6 equivalents of DIPEA, shake well, and pre-activate for 5 minutes. Transfer the activated solution to a synthesis tube, add DMF until the resin is submerged, bubble under nitrogen at room temperature for 60 minutes, and then dry the reaction solution. Wash the resin three times with DMF (10 mL each time). Perform the coupling of Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Arg(Pbf)-OH sequentially using the above method. After the last amino acid is coupled, deprotect Fmoc to obtain a peptide resin with an N-terminal free amino group. After the four amino acids were linked, the resin was washed sequentially with DMF, DCM, and methanol, dried, and 10 mL of pre-chilled lysis buffer (TFA:H2O:TIS = 95:2.5:2.5) was added. The reaction was carried out in an ice bath for 2 hours. The lysis buffer was collected by filtration, and the resin was washed twice with TFA. The filtrates were combined and slowly added dropwise to 10 times the volume of pre-chilled anhydrous diethyl ether. The mixture was stirred to precipitate the peptide, allowed to stand at 4°C for 30 minutes, and centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was washed three times with anhydrous diethyl ether, discarding the supernatant after each centrifugation. The precipitate was dried under vacuum and dissolved in 10% (v / v) acetonitrile aqueous solution. The solution was filtered through a 0.45 μm filter membrane and purified by RP-HPLC (first purification: C18 preparative column, mobile phase A: 0.1% TFA / water, mobile phase B: 0.1% TFA / acetonitrile, gradient elution, collection of the main peak fraction; second purification: 0.1% TFA / acetonitrile). (The TFA system was replaced with an acetic acid / water-acetonitrile system for secondary purification), the fraction was collected, 6% (w / v) mannitol was added, and the mixture was pre-frozen at -40℃ for 3 hours. Then it was dried at -20℃ under vacuum of 10~25 Pa for 20 hours, and then heated to 40℃ for desorption and drying for 6 hours to obtain the active pentapeptide REGRT (purity ≥98%).

[0013] Example 2

[0014] Preparation of butterfly pea flower water extract: Dried butterfly pea flower petals were taken and purified water was added at a material-to-liquid ratio of 1:15 (w / v). The mixture was extracted at 50℃ for 30 minutes. The extract was filtered through a 0.45μm microporous membrane, and the filtrate was collected. The filtrate was concentrated under reduced pressure to a relative density of 1.10 and then freeze-dried at -45℃ and 10~20 Pa to obtain butterfly pea flower extract (moisture content ≤5.0%).

[0015] Example 3

[0016] A corneal damage repair solution containing active pentapeptides: Take 80% volume of water for injection, preheat to 37°C, add 1.0 g / L sodium hyaluronate, allow to swell at room temperature for 8 hours, cool to 20°C, then add 5 mg / L styraxone and 0.05 g / L butterfly pea flower water extract (prepared by the method in Example 2), stir evenly, then add 20 mg / L of the active pentapeptide (REGRT) prepared in Example 1, 20 μg / L of recombinant human nerve growth factor (NGF) and 10 μg / L of recombinant human keratinocyte growth factor-2 sequentially at 15°C, add water for injection to 100% volume, adjust pH to 7.0 ± 0.2, osmotic pressure to 295 mOsmol / kg, filter with a 0.22 μm ultrafiltration membrane for sterilization, and obtain the repair solution.

[0017] Example 4

[0018] A corneal damage repair solution containing active pentapeptides: Take 80% volume of water for injection, preheat to 37°C, add 2 g / L sodium hyaluronate, allow to swell at room temperature for 10 hours, cool to 20°C, then add 8 mg / L styraxone and 0.2 g / L butterfly pea flower water extract (prepared in Example 2), stir evenly, then add 50 mg / L of the active pentapeptide (REGRT) prepared in Example 1, 30 μg / L of recombinant human nerve growth factor (NGF) and 20 μg / L of recombinant human keratinocyte growth factor-2 sequentially at 15~20°C, add water for injection to 100% volume, adjust pH to 7.0±0.2, osmotic pressure to 295 mOsmol / kg, filter with a 0.22 μm ultrafiltration membrane for sterilization, and obtain the repair solution.

[0019] Example 5

[0020] A corneal damage repair solution containing active pentapeptides: Take 80% volume of water for injection, preheat to 37°C, add 3.0 g / L sodium hyaluronate, allow to swell at room temperature for 12 hours, cool to 20°C, then add 10 mg / L styraxone and 0.4 g / L butterfly pea flower water extract (prepared in Example 2), stir evenly, then add 80 mg / L of the active pentapeptide (REGRT) prepared in Example 1, 50 μg / L of recombinant human nerve growth factor (NGF) and 25 μg / L of recombinant human keratinocyte growth factor-2 sequentially at 20°C, add water for injection to 100% volume, adjust pH to 7.0 ± 0.2, osmotic pressure to 295 mOsmol / kg, filter with a 0.22 μm ultrafiltration membrane for sterilization, and obtain the repair solution.

[0021] Comparative Example 1

[0022] Unlike Example 4, this comparative example did not include the active pentapeptide prepared in Example 1.

[0023] Comparative Example 2

[0024] Unlike Example 4, this comparative example did not contain NGF.

[0025] Comparative Example 3

[0026] Unlike Example 4, this comparative example did not contain KGF-2.

[0027] Comparative Example 4

[0028] Unlike Example 4, this comparative example uses an equal amount of Lycium barbarum polysaccharide to replace the butterfly pea flower water extract.

[0029] Test Example 1: Eye Irritation Test

[0030] Six healthy adult New Zealand white rabbits, weighing 2.0–2.5 kg, were selected, consisting of half males and half females. Slit-lamp examination of both eyes was performed 24 hours prior to the experiment. Rabbits with no eye irritation symptoms and negative corneal fluorescein staining were included in the experiment.

[0031] Experimental Groups: Example group: Corneal damage repair solutions containing active pentapeptides prepared in Examples 3-5 of this invention; Reference standard: 0.9% sodium chloride injection (negative control, non-irritating); The left and right sides of the same animal were compared using a self-comparison method. 0.9% sodium chloride injection was instilled into the left eye of each animal as a negative control, while the repair solution prepared in Examples 3-5 was instilled into the right eye. The drugs were administered 50 μL four times a day (simulating the scenario of frequent daily medication in clinical settings) for seven consecutive days.

[0032] Administration procedure: Gently pull down the lower eyelid and drop the medication into the lower conjunctival sac. Passively close the eyelid for about 10 seconds to prevent the medication from spilling out.

[0033] Before each administration and at 1, 24, 48, and 72 hours after the last administration, the degree of eye damage was observed and recorded using a slit-lamp microscope (refer to Appendix A of GB / T 21609-2025, Classification of Eye Damage). At the same time, any abnormal behaviors such as squinting, scratching, or restlessness in the animals during the administration period were also recorded.

[0034] Throughout the administration and observation period, the animals in all groups ate and drank normally, maintained good mental condition, and showed no obvious signs of pain or discomfort such as scratching their eyes, frequent blinking, or restlessness. Slit-lamp examination revealed no abnormalities in the conjunctiva, cornea, or iris in the negative control group and all example groups throughout the experiment. Throughout the entire experimental period, the corneas of all animals remained transparent and free of cloudiness, and the iris texture was clear without swelling.

[0035] In summary, the repair solution of this invention is non-irritating to the conjunctiva, cornea, and iris of normal rabbit eyes, and continuous and frequent administration did not cause corneal epithelial damage, indicating that it has good safety for topical ocular use.

[0036] Experimental Example 2

[0037] The human corneal epithelial cell line (HCE-T) was used to evaluate the cell migration promotion effect of each treatment group using a scratch assay. The experimental groups were: blank control group (PBS), comparative example 1, comparative example 2, comparative example 3, comparative example 4, and example 4 group.

[0038] Experimental methods: (1) HCE-T cells in the logarithmic growth phase were digested with trypsin, resuspended in DMEM / F12 complete medium containing 10% FBS, and the cell density was adjusted to 5×10⁶ cells / year. 5 Cells / mL were seeded into 6-well plates, 2 mL per well. The plates were then incubated at 37°C in a 5% CO2 incubator until a monolayer of 80-90% cell confluence was achieved. (2) Discard the original culture medium and use a sterile 200 μL pipette tip to draw a straight line vertically in the center of each well to form a cell-free scratch area of ​​uniform width. Gently wash twice with PBS to remove any suspended cell debris.

[0039] (3) Add serum-free DMEM / F12 medium containing the corresponding test sample (containing mitomycin C 1 μg / mL to inhibit cell proliferation and eliminate the interference of proliferation effect on migration results) to each well. Each group has 3 replicates.

[0040] (4) At 0 h and 24 h after drug administration, the same scratch field of view was photographed under an inverted phase contrast microscope (40× or 100× magnification). The area of ​​the scratch region at each time point was measured using ImageJ image analysis software. The cell migration rate at 24 h was calculated with the scratch area at 0 h as the baseline (Table 1).

[0041]

[0042] Table 1. 24-hour migration rate (%)

[0043] Table 1 shows that the cell migration rate in the Example 4 group was significantly higher than that in the comparative groups and the blank control group 24 hours after scratching. This indicates that the active pentapeptide REGRT, NGF, KGF-2 and butterfly pea flower water extract can all enhance the migration ability of corneal epithelium. Among them, the active pentapeptide has the greatest impact on the migration ability of corneal cells, and there is a significant synergistic effect among the active ingredients.

[0044] Experiment 3: Rabbit Corneal Epithelial Defect Repair Experiment

[0045] A corneal central epithelial ring scraping defect model was established in New Zealand white rabbits: After anesthesia with 3% sodium pentobarbital (1 mL / kg) via the ear vein, the animals were locally anesthetized with 0.5% promecaine hydrochloride instilled into the ocular surface. An 8.0 mm diameter trephine was used to gently mark the central corneal region. Under a surgical microscope, a corneal epithelial scraper was used to gently scrape away the central corneal epithelium along the marked ring, reaching the Bowman's layer, taking care to avoid damaging the stroma. Immediately after scraping, the area was stained with sodium fluorescein to confirm clear boundaries and no epithelial residue, indicating successful model establishment.

[0046] Postoperatively, 50 μL of ocular drops were administered three times daily (approximately 6 hours apart) until complete corneal epithelial healing. Epithelial healing rate was observed and calculated using sodium fluorescein staining at 0, 12, 24, 36, and 48 hours postoperatively: (initial defect area - remaining defect area) / initial defect area × 100%.

[0047] Animals that successfully developed the model were randomly divided into the following groups according to weight and sex, with 6 animals (6 eyes) in each group: Model control group: 0.9% sodium chloride injection; Positive control group: rhEGF eye drops (Guilin Huanowei Gene Pharmaceutical Co., Ltd.); Example 4 group: Repair solution of Example 4 of this invention; Active pentapeptide group: The active pentapeptide prepared in Example 1 was dissolved alone in water for injection, and the pH was adjusted to 7.0±0.2, and the osmotic pressure was 295mOsmol / kg; Comparative group: Repair solutions prepared by comparative examples 1 to 4.

[0048] Table 2 Epithelial healing rate (%)

[0049] Table 2 shows that the epithelial healing rate of the Example 4 group at each time point was significantly higher than that of the model control groups and the Example groups. This indicates that the active pentapeptide REGRT, NGF, KGF-2, and butterfly pea flower water extract can all promote corneal repair, and none of them can be omitted.

[0050] Experimental Example 4: In Vitro Rabbit Corneal Organ Culture Experiment

[0051] Freshly removed rabbit eyeballs were repeatedly rinsed with antibiotic-containing PBS in a laminar flow hood. The posterior segment of the eyeball tissue was excised 2 mm outside the limbus, preserving the anterior corneal-scleral ring intact. The iris, lens, and ciliary body were carefully removed using microscissors, keeping the intact corneal endothelium facing upwards. The corneal-scleral ring was then transferred to a 6-well plate with the corneal epithelial surface facing upwards. To simulate the mechanical epithelial damage during recipient corneal bed preparation in artificial corneal transplantation, a standard method was used to perform partial epithelial ring scraping in the central corneal region of each group: a 5.0 mm diameter trephine was used to lightly mark the area, and an epithelial scraper was used to evenly scrape away approximately 50% of the epithelium within the marked area (preserving some epithelial islands). This resulted in a non-full-thickness, incomplete epithelial defect in the damaged area, simulating partial epithelial abrasion and reduced activity of the corneal epithelium during surgery.

[0052] After 48 hours of incubation, the surface moisture of the corneas was blotted dry with sterile filter paper, and the wet weight (W1) of each cornea-scleral ring was measured using an analytical balance. The corneas were then dried in an 80°C oven for 48 hours until constant weight, and the dry weight (W2) was measured. The water content was calculated.

[0053] (2) After 48 h of culture, limbal tissue (containing limbal stem cell niches) was harvested and double-stained with calcein-AM (labeling live cells, green fluorescence) and propidium iodide (PI, labeling dead cells, red fluorescence) according to the instructions. After OCT embedding, frozen sections (10 μm thick) were prepared and observed and photographed under a fluorescence microscope. The number of green fluorescent (live cells) and red fluorescent (dead cells) cells per high-power field (400×) was counted using ImageJ, and the viability was calculated.

[0054] Table 3. Corneal water content and limbal epithelial cell survival rate at 48 h for each group

[0055] The water content of the normal corneal stroma remains stable at around 78%. Increased water content indicates exacerbated stromal edema and impaired endothelial pump function or epithelial barrier function. Table 3 shows that the corneal water content in the Example 4 group was not significantly different from that of fresh corneas, while it increased significantly in the model control group. This indicates that the repair solution of this invention can protect the corneal endothelial pump function and prevent stromal edema and epithelial barrier function impairment.

[0056] Data on limbal epithelial cell survival showed that the survival rate of limbal epithelial cells in the example group was significantly higher than that in the model control, indicating that the repair solution of the present invention has a strong protective effect on the limbal stem cell niche, which is crucial for the long-term health maintenance of the recipient's ocular surface after artificial corneal transplantation.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A corneal damage repair solution containing active pentapeptides, characterized in that, The main components include: active pentapeptide 10~100mg / L, recombinant human nerve growth factor NGF 10~50μg / L, recombinant human keratinocyte growth factor-2 KGF-2 5~25μg / L, scutellarin 1~10mg / L, butterfly pea flower water extract 0.05~0.5g / L, and sodium hyaluronate 1.0~3.0g / L; The amino acid sequence of the active pentapeptide is REGRT.

2. The corneal damage repair solution according to claim 1, characterized in that, The preparation method of the butterfly pea flower water extract is as follows: take butterfly pea flower petals, add water at a material-to-liquid ratio of 1g:12~15mL, extract at 45~55℃ for 30~40min, filter, concentrate the filtrate under reduced pressure to a relative density of 1.05~1.10, freeze dry, and obtain butterfly pea flower extract.

3. The corneal damage repair solution according to claim 1, characterized in that, The corneal damage repair solution has a pH of 6.5-7.5, an osmotic pressure of 280-320 mOsm / L, and uses water for injection as the solvent.

4. The method for preparing the corneal damage repair solution according to any one of claims 1 to 3, characterized in that, Includes the following steps: After preheating the water for injection, sodium hyaluronate is added and swelled at 20-30°C for 8-12 hours. Then, scutellarin and butterfly pea flower water extract are added and stirred evenly. At 15-20°C, active pentapeptide, recombinant human nerve growth factor, and recombinant human keratinocyte growth factor-2 are added to adjust the pH and osmotic pressure. The mixture is then filtered to remove bacteria, yielding the corneal damage repair solution.

5. The use of the corneal damage repair solution according to any one of claims 1 to 3 or the corneal damage repair solution prepared by the preparation method according to claim 4 in the preparation of products for corneal damage repair and relief of corneal inflammation.

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