Lachrymatory protein short peptide derivative and application thereof

Abstract

The application provides a lacrimogenic protein short peptide derivative and application thereof, and belongs to the technical field of biological medicines.The lacrimogenic protein short peptide derivative is a SAAAVQGTA short peptide which is polyethylene glycol modified.The lacrimogenic protein short peptide has a persistent effect of promoting tear secretion after being treated by polyethylene glycol.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a short peptide derivative of lacrimal protein and its applications. Background Technology

[0002] Lacritin is a human tear-specific core functional glycoprotein encoded by the LACRT gene. It has a molecular weight of 12.3 kDa and is a highly expressed secreted protein specific to lacrimal gland acinar cells.

[0003] Downregulation of lacritin expression, structural inactivation, and pathway blockade directly lead to insufficient tear production, damage to the ocular surface barrier, neurodegeneration, and chronic inflammation, and are centrally associated with a variety of eye diseases. The most critical indication is aqueous-deficient dry eye syndrome, a globally prevalent eye disease with a prevalence of 5% to 25% in the population. Its pathogenesis includes autoimmune attack, age-related regression, and inflammatory damage to the lacrimal glands. Wearing contact lenses, in particular, can more easily trigger dry eye symptoms.

[0004] Based on the physiological functions and clinical needs of lacritin, global research and development has been conducted on recombinant natural proteins, modified active short peptides, and long-acting eye drop formulations. Among these, the core candidate drug is Lacripep (a short peptide for lacrimal stimulation), a synthetic peptide with a truncated C-terminal active functional domain of natural lacritin. It overcomes the shortcomings of full-length proteins, such as easy degradation, poor stability, and short in vivo half-life. Its mechanism of action differs from existing drugs; it can directly replenish endogenously deficient lacrimal activity through topical eye drops, promoting physiological lacrimal gland secretion, specifically regenerating corneal sensory nerves, rebuilding the neuro-lacrimal gland feedback axis, repairing the corneal epithelial barrier, inhibiting chronic ocular surface inflammation, and stabilizing the tear film. Furthermore, it does not rely on cholinergic neural pathways and is effective for both lacrimal atrophy-related and Sjögren's syndrome-related dry eye. However, compared to full-length lacrimal proteins, short peptides for lacrimal stimulation face challenges such as poor activity and difficulty in screening.

[0005] Based on this, the present invention is proposed. Summary of the Invention

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a short peptide derivative of lacrimal protein, wherein the short peptide derivative of lacrimal protein is a polyethylene glycol-modified SAAAVQGTA short peptide.

[0007] The present invention also provides a drug for promoting tear secretion, the drug containing the lacrimal protein short peptide derivative.

[0008] The present invention also provides the application of the aforementioned lacrimal protein short peptide derivative in the preparation of a drug that promotes tear secretion.

[0009] The present invention also provides the use of the lacrimal protein short peptide derivative in the preparation of a medicament for treating dry eye syndrome.

[0010] The present invention also provides the application of the aforementioned tear-inducing protein short peptide derivative in the preparation of eye drops for relieving dry eye symptoms after wearing contact lenses.

[0011] The present invention also provides an application of the aforementioned tear-inducing protein short peptide derivative as a contact lens soaking solution.

[0012] This invention extracts a short peptide (SAAAVQGTA, as shown in SEQ ID NO: 2) from the lacrimal protein sequence (as shown in SEQ ID NO: 1), and experimentally verifies that the short peptide, after being modified with polyethylene glycol, has the effect of promoting tear secretion, and its stability and persistence are superior to the long lacrimal protein shown in SEQ ID NO: 1. Detailed Implementation

[0013] 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.

[0014] Example 1

[0015] Tear-inducing protein (SEQ ID NO: 1): EDASSDSTGADPAQEAGTSKPNEEISGPAEPASPPETTTTAQETSAAAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQKLLKKFSLLKPWA.

[0016] The aforementioned tear-inducing protein is a long protein sequence that has been verified to have tear-inducing effects. To address the problems of easy degradation, poor stability, and poor persistence of long tear-inducing protein sequences, this invention screened a short peptide sequence from the aforementioned sequence and modified it to obtain a short peptide derivative of the tear-inducing protein. This short peptide derivative of the tear-inducing protein exhibits superior tear-inducing effects compared to the long tear-inducing protein sequence, and also possesses higher stability and persistence.

[0017] The short peptide sequence obtained by screening in this invention is: SAAAVQGTA (SEQ ID NO: 2).

[0018] First, the effects of long and short peptides of lacrimal protein on promoting tear secretion were determined using the Hillmer test strip method. The specific experimental procedure is as follows: (1) Laboratory animals Six-week-old male Westa rats were used. Each animal was housed for one week at room temperature of 23±2℃, humidity of 50±5%, and a 12-hour day / night cycle (day: 07:00-19:00) before being used in experiments. Animals had free access to food and water during both housing and experimental periods.

[0019] (2) Experimental grouping

[0020] The experiment consisted of three groups: a control group, a long peptide group, and a short peptide group, with five animals in each group. The long and short peptide groups received peptide-containing eye drops prepared with physiological saline at a concentration of 5 μmol / kg. The control group received an equal volume of physiological saline. Both the long and short peptides were synthesized using conventional solid-phase synthesis methods.

[0021] (3) Test

[0022] Ten minutes after administration, a Hillmer test strip (2 mm wide) was placed on the lower eyelid, and the length of tear saturation (in mm) was measured after 5 minutes. The results are shown in Table 1. It is evident that the selected short peptide had a poor lacrimal effect.

[0023] Table 1. Tear wetting length (mm) in different treatment groups

[0024] The short peptide was then modified using the following strategy: Strategy 1: Polyglycolic Short Peptides Accurately weigh SAAAVQGTA lyophilized powder (purity ≥90%), dissolve it in 0.1M sodium bicarbonate buffer (pH 7.5), vortex to mix, sonicate to aid dissolution, and let stand at room temperature (25℃) for 5 min to ensure complete dissolution without precipitation, thus preparing a 1 mg / mL short peptide solution.

[0025] Weigh out methoxy polyethylene glycol active ester (mPEG-NHS) powder (5kDa) and prepare a 10 mg / mL mPEG-NHS-DMSO solution using anhydrous DMOS. Prepare and use immediately.

[0026] At a molar ratio of short peptide to mPEG-NHS of 1:2.5, mPEG-NHS-DMSO solution was slowly added dropwise to the short peptide solution while gently mixing, stabilizing the pH between 7.4 and 7.6. The reaction was carried out at room temperature (25°C) in the dark with magnetic stirring for 2 h. After the reaction reached the endpoint, 50 mM glycine was added and stirred at room temperature for 15 min to quench all free active NHS groups in the system, thus terminating the coupling reaction.

[0027] The reaction solution was ultrafiltered three times by centrifugation at 4000 r / min using a 10 kDa ultrafiltration tube, with each resuspending after dilution with ultrapure water. Unreacted mPEG, glycine, NHS byproducts, and DMSO were removed, while PEGylated short peptides were retained. After purification by reversed-phase high-performance liquid chromatography (RP-HPLC) (purity ≥90%), the PEGylated short peptides were concentrated and lyophilized to obtain the PEGylated short peptides.

[0028] Strategy 2: Methionized short peptides

[0029] Accurately weigh SAAAVQGTA lyophilized powder (purity ≥90%) and add it to a dry round-bottom flask. Purge the flask with dry nitrogen for 5 min to ensure an anhydrous reaction environment. Add anhydrous methanol to prepare an 8 mg / mL short peptide solution, and stir magnetically until completely dissolved and clear.

[0030] An anhydrous methanol to thionyl chloride solution was prepared by slowly adding thionyl chloride dropwise to anhydrous methanol under ice bath conditions, with a volume ratio of 20:1 (anhydrous methanol: thionyl chloride = 20:1).

[0031] The acidic methanol solution was completely transferred to the short peptide solution, the reaction flask was sealed, and the reaction was carried out under nitrogen protection at room temperature (25°C) with stirring for 6 hours in the dark. After the reaction was completed, the methanol, excess thionyl chloride, and generated HCl gas were evaporated at low temperature in a 40°C water bath until no acidic odor remained. Crude methyl esterified short peptides were obtained.

[0032] The crude product was dissolved in a small amount of anhydrous dichloromethane and slowly added dropwise to ice-cold anhydrous diethyl ether (volume ratio 1:15), causing the short peptide esterification to precipitate. The precipitate was collected by centrifugation and washed three times with ice-cold diethyl ether to remove residual catalyst and small molecule impurities. The ether was removed by vacuum drying to obtain the dried crude peptide. The peptide was purified by RP-HPLC (purity ≥90%), concentrated, and lyophilized to obtain the methyl esterified short peptide.

[0033] Strategy 3: Cyclic Short Peptides

[0034] Accurately weigh SAAAVQGTA lyophilized powder (purity ≥90%) and dilute it with a large amount of anhydrous DMF, strictly controlling the final concentration to 1 mmol / L (to maximally inhibit intermolecular cross-linking polymerization and only allow intramolecular cyclization to occur). Seal the solution under nitrogen atmosphere and stir at room temperature (25°C) until the short peptide is completely dissolved and clear. Add 2.0 eq DIPEA (molar ratio: short peptide: DIPEA = 1:2) to the short peptide solution.

[0035] Under ice bath conditions, the condensation system was prepared with HBTU: 1.0 eq and HOBt: 1.0 eq (molar amounts are relative to short peptides).

[0036] The reaction system was placed in an ice bath at 0°C. The HBTU / HOBt mixed powder was dissolved in a small amount of anhydrous DMF and slowly added dropwise to the short peptide solution. After the addition was complete, the system was sealed and protected with nitrogen. The reaction was stirred at 0°C for 2 h, then allowed to rise naturally to room temperature (25°C) and continued for 12 h. After the reaction was complete, a small amount of ultrapure water was added to quench the remaining active condensation intermediate, and the reaction was terminated by standing at room temperature for 10 min. Then, the reaction was carried out under reduced pressure and at low temperature (≤35°C) to remove most of the DMF. The remaining viscous residue was reconstituted with a small amount of acetonitrile-water for purification. The residue was purified by RP-HPLC (purity ≥90%), concentrated, and lyophilized to obtain the cyclized short peptide.

[0037] The short peptide sequence SAAAVQGTA of this invention contains only an N-terminal free amino group and a C-terminal free carboxyl group, and lacks active side chain functional groups such as lysine, aspartic acid, and glutamic acid. Therefore, the PEGylation, C-terminal methyl esterification, and proto- and proto-lactam cyclization modification sites are all unique, and there are no multi-site secondary modifications or structural isomers.

[0038] The tear wetting length of different modified short peptides was measured according to the aforementioned method to evaluate the effect of different modification methods on the tear secretion-promoting function of the short peptides. The results are shown in Table 2. It can be seen that polyethylene glycolation and cyclization of the short peptides can significantly promote tear secretion.

[0039] Table 2. Tear wetting length (mm) of different modified short peptides

[0040] Example 2

[0041] The stability and durability of the preferred polyethylene glycol-modified and cyclized short peptides from Example 1 were tested, with the long peptide as a control and physiological saline as a blank control.

[0042] Test method: After administering the drug according to the method in Example 1, the tear wettability length of the Hillmer test strip was measured at certain time intervals, and the statistical results are shown in Table 3.

[0043] Table 3. Durability test of long peptides and short peptides with different modifications (mm)

[0044] As shown in Table 3, the tear wetting length in the blank control group remained stable at around 10 mm. The duration of action of the cyclized short peptide was between 15 and 30 minutes, that of the long peptide was between 30 minutes and 1 hour, while that of the PEGylated short peptide exceeded 2 hours. This indicates that the PEGylated short peptide SAAAVQGTA not only significantly improved the tear secretion promotion effect but also became more stable and longer-lasting.

[0045] As can be seen from the above embodiments, the present invention provides a short peptide derivative of lacrimal protein, which has a long-lasting effect of promoting tear secretion after being treated with polyethylene glycol.

[0046] 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 short peptide derivative of a lacrimal-inducing protein, characterized in that, The tear-inducing protein short peptide derivative is a polyethylene glycol-modified SAAAVQGTA short peptide.

2. A drug that promotes tear secretion, characterized in that, The drug contains the lacrimal protein short peptide derivative as described in claim 1.

3. The use of the lacrimal protein short peptide derivative of claim 1 in the preparation of a drug that promotes tear secretion.

4. The use of the lacrimal protein short peptide derivative of claim 1 in the preparation of a medicament for treating dry eye syndrome.

5. The use of the lacrimal protein short peptide derivative of claim 1 in the preparation of eye drops for relieving dry eye symptoms after wearing contact lenses.

6. The use of the tear-inducing protein short peptide derivative of claim 1 as a contact lens soaking solution.

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