New uses of 4f dimeric polypeptides and their lipidated versions

Abstract

The application discloses a new use of 4F dimer polypeptide and lipids thereof, and the 4F dimer polypeptide and lipids thereof are used for preparing a drug for treating dry eye syndrome. The application has the advantages that the application innovatively finds that pHDL has very good effects on dry eye syndrome, and has the advantages of synergistic treatment for the multi-factor pathological mechanism of dry eye syndrome. Therefore, compared with the existing single mechanism drug (such as cyclosporine eye drops for only inhibiting inflammation), the multi-dimensional action mechanism of pHDL precisely matches the complex pathological network of dry eye syndrome, and is expected to improve the coverage range of the curative effect on refractory dry eye syndrome.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a new use of 4F dimer polypeptide and its lipidated product. BACKGROUND

[0002] Dry eye disease (DED) is a complex disease caused by multiple pathological mechanisms such as tear quality or quantity abnormalities, ocular surface inflammatory response, neural regulation imbalance and environmental factors. The etiology of DED can include autoimmune diseases (such as Sjogren's syndrome), meibomian gland dysfunction (MGD), lacrimal gland secretion deficiency, oxidative stress damage and corneal epithelial barrier destruction. At present, the global incidence of DED is as high as 5%-50%, and it is showing a significant upward trend with the increase of electronic device use, the aggravation of aging and the increase of environmental pollution. Existing treatment methods such as artificial tears, local anti-inflammatory drugs (such as cyclosporine A, glucocorticoids) or mucin secretion promoters (such as diquafosol sodium) can partially alleviate symptoms, but they are mostly aimed at a single pathological link and are difficult to comprehensively intervene in the process of DED mediated by multiple factors. For example, although traditional artificial tears can temporarily lubricate the ocular surface, they have poor tear film stability, high viscosity formula leading to blurred vision, lack of lipid layer repair ability and other limitations; and long-term use of anti-inflammatory drugs may cause corneal toxicity or systemic side effects. In view of the difficulty of single target treatment strategy to cover the pathological link of DED, the development of a multi-effect therapy that can simultaneously regulate inflammation, repair ocular surface barrier, supplement tear film lipid and prolong ocular surface residence time has become an urgent need in the field of dry eye treatment.

[0003] High-Density Lipoprotein (HDL) is the smallest and densest lipoprotein in all plasma lipoproteins in the human body, with core functions of anti-inflammatory, anti-oxidation and lipid transport regulation, and is a research hotspot for the treatment of cardiovascular diseases. Apolipoprotein A-I (ApoA-I) is the most important structural and functional component of HDL, and the ester formed by its self-assembly with phospholipids can simulate the various biological functions of natural HDL. However, the cost of high-purity ApoA-I full-length protein is too high, the acquisition process is complex, the production cycle is long, and it is difficult to be widely used in the laboratory or clinically promoted. 4F polypeptide and its dimer are efficient substitutes for ApoA-I full-length protein, with low cost, easy mass production and avoidance of the immunogenicity of ApoA-I full-length protein. Through microfluidic technology, 4F dimer polypeptide can be mixed with neutral ion phospholipid DMPC to self-assemble into biomimetic HDL with a disc-shaped diameter of about 30 nanometers, which is called pHDL. Compared with natural HDL, pHDL retains the biological functions of natural HDL while having unique advantages of low immunogenicity, high tissue permeability and targeted delivery. Among them, DMPC is highly similar to the natural tear film lipid (such as the phospholipid secreted by the meibomian gland), which can integrate into the tear film lipid layer and fill the defect area. At the same time, DMPC can form an ordered arrangement of lipid bilayers, reduce tear evaporation, improve tear film stability, and alleviate the core pathophysiological characteristics of dry eye syndrome - hyperosmolarity and excessive evaporation of the tear film, thereby stabilizing and repairing the tear film lipid layer. In particular, DMPC as an electrically neutral phospholipid can also avoid the potential irritability of cationic components to the corneal epithelium, and is suitable for long-term use. As an analogue of ApoA-I, 4F dimer polypeptide has multiple anti-inflammatory and anti-oxidation functions, and can also play the cholesterol reverse transport function of ApoA-I, participate in the regulation of meibomian gland lipid metabolism, improve the quality of lipid secretion, and reduce the occurrence of obstructive dry eye.

[0004] Therefore, how to apply pHDL in the treatment of dry eye is a problem to be solved by those skilled in the art. SUMMARY

[0005] The purpose of the present application is to solve the above technical problems.

[0006] In order to achieve the above object, the technical scheme adopted by the present application is as follows: The application provides a new use of 4F dimer polypeptide and lipidated product thereof, and the 4F dimer polypeptide and lipidated product thereof are used for preparing a drug for treating dry eye disease. Dry eye disease (DED) is a complex disease caused by multiple pathological mechanisms such as tear quality or quantity abnormalities, ocular surface inflammatory response, neural regulation imbalance and environmental factors. The etiology of DED can cover autoimmune diseases (such as Sjogren's syndrome), meibomian gland dysfunction (MGD), lacrimal gland secretion deficiency, oxidative stress damage and corneal epithelial barrier destruction. At present, the global incidence of DED is as high as 5%-50%, and it shows a significant upward trend with the increase of electronic device use, the aggravation of aging and the increase of environmental pollution. The existing treatment methods such as artificial tears, local anti-inflammatory drugs (such as cyclosporine A, glucocorticoids) or mucin secretion promoters (such as diquafosol sodium) can partially relieve symptoms, but they are mostly aimed at a single pathological link and are difficult to comprehensively intervene in the process of DED mediated by multiple factors. For example, although traditional artificial tears can temporarily lubricate the ocular surface, they have limitations such as poor tear film stability, blurred vision caused by high viscosity formula, lack of lipid layer repair ability, etc.; and long-term use of anti-inflammatory drugs may cause corneal toxicity or systemic side effects. In view of the difficulty of single target treatment strategy to cover the pathological links of DED, it is an urgent need in the field of dry eye treatment to develop a multi-effect therapy that can simultaneously regulate inflammation, repair ocular surface barrier, supplement tear film lipid and prolong ocular surface residence time.

[0007] High-Density Lipoprotein (HDL) is the smallest and densest lipoprotein in all plasma lipoproteins in the human body, with core functions of anti-inflammatory, antioxidant and lipid transport regulation, and is a research hotspot for the treatment of cardiovascular diseases. Apolipoprotein A-I (ApoA-I) is the most important structural and functional component of HDL, and the ester formed by its self-assembly with phospholipids can simulate the multiple biological functions of natural HDL. However, the cost of high-purity ApoA-I full-length protein is too high, the acquisition process is complex, the production cycle is long, and it is difficult to be widely used in the laboratory or clinically promoted. 4F polypeptide and its dimer are efficient substitutes for ApoA-I full-length protein, with low cost, easy mass production and avoidance of the immunogenicity of ApoA-I full-length protein. Through microfluidic technology, 4F dimer polypeptide is mixed with neutral ion phospholipid DMPC to self-assemble into biomimetic HDL with a diameter of about 30 nanometers in the form of a flat disc, which is called pHDL. Compared with natural HDL, pHDL retains the biological functions of natural HDL, while has the unique advantages of low immunogenicity, high tissue permeability and targeted delivery. At present, pHDL is mainly used in cardiovascular diseases, and the inventors have innovatively found that pHDL has very good effect on dry eye syndrome, for example: the synergistic treatment advantage for the multi-factor pathological mechanism of dry eye syndrome. In terms of anti-inflammatory and immune regulation, 4F polypeptide can block the inflammatory cascade. In terms of antioxidant stress protection, the phospholipid bilayer structure can efficiently capture free radicals and degrade oxidized lipids through the peroxidase activity of phosphatidylcholine to repair the oxidative damage of corneal epithelium. In terms of tear film lipid layer repair: DMPC can supplement the missing polar lipids in patients with meibomian gland dysfunction, improve the stability of tear film and the balance of osmotic pressure. Therefore, compared with existing single-mechanism drugs (such as cyclosporine eye drops which only inhibit inflammation), the multi-dimensional action mechanism of pHDL precisely fits the complex pathological network of dry eye syndrome, and is expected to improve the efficacy coverage of refractory dry eye syndrome.

[0008] Further, the gene sequence of the 4F dimer polypeptide is shown as SEQ ID NO: 1. The 4F dimer polypeptide and the lipidate thereof are a complex of the 4F dimer polypeptide and phospholipid, and the molar ratio of the 4F dimer polypeptide and the phospholipid is 1:50-1:100. The conventional HDL analogues have poor corneal penetration due to large molecular weight, and it is difficult to meet the needs of ocular surface administration. In terms of the particle size of the drug molecules, under normal physiological conditions, the intercellular space is about 20-30 nanometers, and the barrier function is complete to prevent pathogens and harmful substances from penetrating; and under the condition of dry eye, due to apoptosis, degradation of connecting proteins or edema, the space can be expanded to more than 50 nanometers, resulting in barrier damage and increased permeability. By using microfluidic technology to prepare pHDL nanoparticles with a particle size of about 30 nanometers, the corneal penetration efficiency can be significantly improved. In addition, the nanostructure can also improve the ocular surface retention time, avoiding the defect that the conventional eye drops are easily washed away by tears. In terms of charge: the use of neutral ion phospholipid can reduce local irritation, and also can be integrated into the tear film lipid layer to fill the damaged area and prolong the ocular surface retention time. Therefore, the pHDL eye drops are superior to the conventional preparations in terms of bioavailability, local tolerance and administration frequency, and no safety risks are found.

[0009] In addition, the differentiated design of pHDL provides an opportunity for clinical transformation. In terms of drug composition, the ApoA-I analogue polypeptide 4F dimer (sequence as SEQ ID NO: 1) is combined with a specific ratio (molar ratio 1:50-1:100) of natural neutral phospholipid to give pHDL with dual optimization of anti-inflammatory and antioxidant and tear film integration. In terms of preparation process: the "one-step self-assembly microfluidic" preparation process breaks through the defects of low yield and unstable batch of traditional methods, and can realize large-scale production.

[0010] Further, the 4F dimer polypeptide and the lipidate thereof as a drug carrier can also load drugs when preparing a drug for treating dry eye. The 4F dimer polypeptide and the lipidate thereof are prepared by using physiological saline, the concentration of the 4F dimer polypeptide is 5-20 mg / ml, and the concentration of the lipidate is 5-20 mg / ml.

[0011] In summary, by using the above technical solutions, the present application has the following beneficial effects: the present application innovatively finds that pHDL has very good effect for dry eye, and has the synergistic treatment advantage for the multi-factor pathological mechanism of dry eye. Therefore, compared with the existing single mechanism drug (such as cyclosporine eye drops which only inhibit inflammation), the multi-dimensional action mechanism of pHDL precisely fits the complex pathological network of dry eye, and is expected to improve the coverage of refractory dry eye. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 pHDL morphology under transmission electron microscope.

[0013] Figure 2 Ocular index of dry eye after 3 days of intervention in mouse atropine model; in the figure:*** p <0.005 vs Blank., p <0.05, and *** p <0.01, and **** p <0.005 vs DES.

[0014] Figure 3 Ocular index of dry eye after 7 days of intervention in mouse atropine model; in the figure:*** p <0.005 and **** p <0.001 vs Blank., p <0.05, and *** p <0.005 vs DES.

[0015] Figure 4 HE staining of cornea and lacrimal gland of mouse atropine dry eye model.

[0016] Figure 5 Safety index of mouse atropine dry eye model.

[0017] Figure 6 Ocular index after 5 days of intervention in mouse benzalkonium chloride dry eye model; in the figure,* p <0.05 and *** p <0.005 vs Blank., p <0.05 and *** p <0.005 vs DES.

[0018] Figure 7 Ocular index after 10 days of intervention in mouse benzalkonium chloride dry eye model; in the figure,*** p <0.005 vs Blank., p <0.005 vs DES.

[0019] Figure 8 HE staining of cornea and lacrimal gland of mouse benzalkonium chloride dry eye model.

[0020] Figure 9 PAS staining of conjunctiva of mouse benzalkonium chloride dry eye model.

[0021] Figure 10This serves as a safety indicator for the mouse benzalkonium chloride dry eye model. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0023] Example: This invention discovers a novel use for 4F dimer polypeptides and their lipid derivatives in the preparation of drugs for treating dry eye syndrome. The gene sequence of the 4F dimer polypeptide is shown in SEQ ID NO:1. The 4F dimer polypeptide and its lipid derivatives are complexes of the 4F dimer polypeptide and phospholipids. The molar ratio of the 4F dimer polypeptide to phospholipids is 1:50. It should be noted that the molar ratio of the 4F dimer polypeptide to phospholipids can also be 1:100, 1:60, 1:70, 1:80, or 1:90; these molar ratios are all acceptable. The particle size of the 4F dimer polypeptide and its lipid derivatives, which are complexes of the 4F dimer polypeptide and phospholipids, is 30 nm. The 4F dimer peptide and its esterified form (pHDL) were prepared as follows: (1) Preparation of aqueous phase: 20 mg of L-4F peptide (DWFKAFYDKVAEKFKEAFPDWFKAFYDKVAEKFKEAF) in C-terminal amidated dimer form was accurately weighed, dissolved in sterile water, and thoroughly mixed before use. (2) Preparation of organic phase solution: 50 mg of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was accurately weighed, dissolved in anhydrous ethanol, and thoroughly vortexed before use. pHDL was synthesized at a flow rate of 1 mL / min for the organic phase and 5 mL / min for the aqueous phase. The synthesized nanoparticles were concentrated by centrifugation at 4000 ×g for 15 minutes at 25°C using a 10 kD ultrafiltration tube, and then centrifuged three more times under the same centrifugation conditions with ddH2O added to wash away residual ethanol. After concentration and washing, store at 4°C. It should be noted that the molar ratio of 4F dimer peptide to phospholipid can also be 1:100, 1:60, 1:70, 1:80, or 1:90, with consistent effects. When used as a treatment for dry eye, it also includes the drugs it can encapsulate as a drug carrier. The 4F dimer peptide and its lipid derivative are prepared using physiological saline, with a 4F dimer peptide concentration of 5-20 mg / ml and a lipid derivative concentration of 5-20 mg / ml. The obtained F dimer peptide and its lipid derivative (pHDL) are shown below. Figure 1TEM observation of the morphology of pHDL: 10 μΐ of freshly prepared pHDL nanoparticles were dropped onto a parafilm film to form a droplet. The front side of the carbon supported copper grid was treated with the droplet for 2 min, and then the copper grid was further treated with a 2% phosphotungstic acid droplet for 1 min. The residual staining solution on the surface of the copper grid was absorbed with a wet filter paper, and the copper grid was placed in a sample box with the front side facing up to dry overnight. The next day, the blank carrier and emodin nanoparticles were observed using a transmission electron microscope at an acceleration voltage of 80 kV.

[0024] The following illustrates two models that 4F dimer peptides and their lipidated products can be used to treat dry eye.

[0025] 1. The first part of the efficacy evaluation: The function of 4F dimer peptides and pHDL in restoring tear secretion was evaluated by using atropine to model the blockade of cholinergic M3 receptors to inhibit lacrimal gland secretion function, simulating a model of DED caused by water deficiency. A 1% atropine (1 mg / 100 mg) was used to model DED, and the amount of eye drops was 5 μΐ per eye. The modeling time was 8 and 14 o'clock, and the administration time was 11 and 17 o'clock. After 7 days of DED modeling using atropine eye drops, 0.1% sodium hyaluronate eye drops were administered as a positive drug. The treatment groups were divided into low-dose (5 mg / mL), medium-dose (10 mg / mL), and high-dose (20 mg / mL) peptides and low-dose (5 mg / mL), medium-dose (10 mg / mL), and high-dose (20 mg / mL) pHDL interventions. On the 3rd and 7th days of modeling and administration, the tear secretion test (SIT) and corneal fluorescein staining score (FLS) of each group of mice were detected. The samples were taken the day after the last test to evaluate the safety and efficacy of the drugs.

[0026] 1.1 Alleviate dry eye symptoms (Figures 2, 3): evaluated by tear secretion test and tear film stability test. Among them, Schirmer test can directly reflect the functional status of lacrimal gland, which is the core index of tear secretion function and water deficiency. The amount of tear secretion in each group of mice was detected by phenol red cotton thread experiment. The mice were fixed, and the phenol red cotton thread was clamped with an ophthalmic microforceps and placed in the conjunctival sac of the lower 1 / 3 of the mouse eyelid. After 15 seconds, it was taken out and the wet length of the cotton thread was measured with a vernier caliper, which was the length of the cotton thread changed from yellow to red, accurate to 0.1 mm. The average value of both eyes was taken. Secondly, the tear film stability was detected by corneal fluorescein staining score. Corneal fluorescein staining score is used to directly reflect the damage of ocular surface epithelial barrier and secondary inflammatory injury caused by dry eye, which is a key indicator for evaluating disease progression and treatment effect. The mice were fixed, and the fluorescein sodium detection test paper was moistened with 1 drop of normal saline. The tip of the test paper was gently touched on the inner side of the lower eyelid of the mouse and stayed for a while. The fluorescence was evenly distributed by touching the eyelid a few times. The corneal fluorescence staining was observed under cobalt blue light with a slit lamp, and the staining grade was evaluated. The cornea was divided into 4 quadrants, and no staining was scored as 0. Staining was scored as light, medium, and heavy, with 1 point for less than 5 points, 3 points for block staining or filamentous material, and 2 points for between the two. A total of 0-12 points.

[0027] 1.1.1 After continuous administration for 3 days (Figure 2), Figure 2 compared with the blank group, the tear secretion of the model DED group was significantly reduced, and the corneal epithelium had signs of damage. Compared with the model group, after treatment with the positive drug sodium hyaluronate eye drops (SH), there was no significant improvement in tear secretion and corneal epithelial damage. After intervention with different doses of 4F dimer peptide, there was a trend of improvement in tear secretion and corneal epithelial damage, with the most significant improvement in corneal damage at high dose (20 mg / mL). After intervention with different doses of pHDL, the high dose group (20 mg / mL) had a significant improvement in tear secretion and corneal epithelial damage.

[0028] 1.1.2 After continuous administration for 7 days (Figure 3), Figure 3 compared with the blank group, the tear film break-up time of the model group was significantly shortened, and the corneal epithelium was severely damaged. Compared with the model group, after treatment with the positive drug sodium hyaluronate eye drops (SH), there was no significant improvement in tear secretion and corneal epithelial damage. After intervention with different doses of 4F dimer peptide and pHDL, there was a trend of improvement in tear secretion, with the most significant improvement at high dose (20 mg / mL); different doses of 4F dimer peptide and pHDL had a significant effect on corneal damage, with the best effect at high dose.

[0029] 1.2 Improve the histopathology of dry eye (Figure 4), Figure 4): After being sacrificed, the left eyeballs of the mice were taken, fixed with 4% paraformaldehyde solution for 24 h, embedded with paraffin, and cut into sections with a thickness of about 4 μm. After being dyed and dried, the changes in the histopathological structures of the corneas and lacrimal glands were observed under an optical microscope, and images were collected for statistical analysis of the thickness of the corneal epithelium. The results showed that

[0030] The hematoxylin and eosin (HE) staining of the corneas and lacrimal glands of the mice in each group showed that Figure 4 The normal group of mice had a normal corneal tissue structure, regular corneal epithelial cell morphology, clear hierarchical structure, clear outline, uniform staining, and tight cell-cell connection. The epithelial cells were arranged in an orderly manner in about 3-4 layers, and the surface was smooth. No thickening was observed. The collagen fiber plate layer parallel to the cornea formed the stroma layer, and the distributed fibroblasts were arranged tightly in the stroma layer. No inflammatory cell infiltration was observed in the tissue. Compared with the normal group, the model group showed a non-smooth surface of the corneal epithelial layer, disordered cell arrangement, and epithelial cell shedding, as indicated by the red arrows. The collagen fibers in the stroma layer were loosely arranged, and the cell-cell connection was slightly loose, as indicated by the blue arrows. Compared with the model group, the SH group had a more orderly arrangement of corneal epithelial cells, but the cell membrane boundary was blurred, and the cytoplasm was partially edematous and lightly stained. The peptide dose groups showed slight improvement in corneal damage, but there were still rough epithelial layers, cell shedding, disordered cell arrangement, and loose collagen fibers. The peptide 20 mg group showed more obvious recovery. The pHDL 5 mg group had a non-smooth surface of the corneal epithelial layer, obvious cell shedding, and cytoplasmic edema. The collagen fibers in the stroma layer were arranged in an orderly manner. The pHDL 10 mg and 20 mg groups showed some recovery of corneal epithelial damage, and the pHDL 20 mg group showed more obvious recovery. The corneal epithelial surface was smooth, the cell morphology was regular, and the stroma layer was arranged in an orderly manner.

[0031] In addition, for the lacrimal glands, the normal group of mice had lacrimal gland tissue composed of lobules of different sizes, uniform-sized alveoli, regular morphology, tightly arranged alveolar cells, intact lacrimal epithelial cells, and high columnar shape. The cytoplasm was rich in secretory vacuoles, and the alveolar cavity contained vesicular mucus. Compared with the normal group, the model group had atrophic lacrimal gland cells, irregular arrangement, structural damage, reduced mucus in the lumen, and unevenly sized and arranged alveoli and lacrimal epithelium, as indicated by the green arrows. The connective tissue was significantly swollen, and there were inflammatory cell infiltrations in the interstitium. Compared with the model group, the positive drug sodium hyaluronate eye drops (SH) group showed no significant reduction in lacrimal gland tissue damage and atrophic lacrimal epithelial cells. The peptide 5 mg and 10 mg groups showed no obvious improvement in the lacrimal gland structure, and there were inflammatory cell infiltrations. The peptide 20 mg group had slightly regular lacrimal gland cells. The pHDL 5 mg group had disordered alveolar arrangement. The pHDL 10 mg and 20 mg groups showed reduced lacrimal gland histopathological damage, complete lacrimal gland structure, and regular lacrimal epithelial cell morphology.

[0032] 1.3 Safety (ALT, AST, TP, ALB, LDH, CK, BUN, CR) Figure 5 ): Serum biochemical tests were performed on mice after atropine modeling, 4F dimer polypeptide, and pHDL intervention. No intervention scheme was found to cause damage to the liver and kidney of mice. ALT: glutamic-pyruvic transaminase; AST: glutamic-oxaloacetic transaminase; TP: total protein; ALB: albumin; LDH: lactate dehydrogenase; CK: creatine kinase; BUN: serum urea nitrogen; CR: serum creatinine.

[0033] 2. Pharmacodynamic evaluation part II: The use of benzalkonium chloride (BAC) to model the destruction of the ocular surface epithelial barrier, induce inflammatory response and tear film instability can cause lipid layer defects, ocular surface inflammation and epithelial damage, simulate hyper-evaporation type dry eye or mixed dry eye model. 0.2% benzalkonium chloride eye drops were used to model DED, and the modeling time was 8 and 14 o'clock, and the administration time was 11 and 17 o'clock. After 9 days of benzalkonium chloride eye drops for DES modeling, 0.1% sodium hyaluronate eye drops were used as positive drug, and the treatment group pHDL low dose (5 mg / mL), medium dose (10 mg / mL) and high dose (20 mg / mL) were intervened for 10 days. After the observation of tear film break-up time (BUT) and corneal fluorescein staining score (FLS) of each group of mice, the samples were taken the next day for detection to evaluate the safety and efficacy of the drug.

[0034] 2.1 Alleviating dry eye symptoms (BUT, FLS) Figure 6 , 7 ): Evaluated by tear film break-up time observation and tear film stability test.

[0035] Among them, the tear film break-up time observation (break-up time, BUT) detects the stability of the tear film by detecting the tear film break-up time. The tear film break-up time can reflect: (1) abnormal lipid layer, i.e. meibomian gland dysfunction (MGD) causes insufficient secretion of lipids, which cannot form a stable tear film, and the evaporation rate is accelerated; (2) mucous layer defect, i.e. reduction of goblet cells (such as vitamin A deficiency, chronic inflammation) destroys the adhesion of the tear film; (3) high osmotic tear, i.e. the osmotic pressure increases after the evaporation and concentration of the tear, further destroying the stability of the tear film, to suggest abnormality of the meibomian gland function or tear film composition. Fix the mouse, wet the fluorescein sodium test paper with 1 drop of normal saline, gently touch the inner lower eyelid of the mouse with the tip of the test paper and stay for 10 s, and help blink 3 times to make the fluorescein evenly distributed. Under the cobalt blue light of the slit lamp, observe the time when the first black dry spot appears on the cornea. Take the average of both eyes. Secondly, the stability of the tear film is detected by the corneal fluorescein staining score. The corneal fluorescein staining score is used to directly reflect the damage of the ocular surface epithelial barrier and secondary inflammatory injury caused by dry eye syndrome, and is a key indicator for evaluating disease progression and treatment effect. The stability of the tear film is detected by the corneal fluorescein staining score. Fix the mouse, wet the fluorescein sodium test paper with 1 drop of normal saline, gently touch the inner lower eyelid of the mouse with the tip of the test paper and stay for a moment, and help blink a few times to make the fluorescein evenly distributed. Under the cobalt blue light of the slit lamp, observe the corneal fluorescence staining and evaluate the staining grade. The scoring method is to divide the cornea into 4 quadrants, and no staining is scored as 0, staining is scored as light, medium and heavy, 1 point is less than 5 points, 3 points is block staining or filamentous, 2 points is between the two, a total of 0-12 points.

[0036] 2.1.1 After 5 days of continuous administration (Day 5) Figure 6 ), compared with the blank group mice, the tear film break-up time of the model DED group mice was shortened, and the corneal epithelial damage was severe. Compared with the model group, after treatment with the positive drug sodium hyaluronate eye drops (SH), the tear film break-up time was significantly improved, and the corneal damage had a tendency to improve. Among the different dose pHDL treatment groups, the tear film break-up time of the mice in the medium dose group (10 mg / mL) and the high dose group (20 mg / mL) was significantly prolonged after treatment, and the corneal damage was improved.

[0037] 2.1.2 After 10 days of continuous administration (Day 10) Figure 7 ), compared with the blank group mice, the tear film break-up time of the model group mice was significantly shortened, and the corneal epithelial damage was severe. Compared with the model group, after treatment with the positive drug sodium hyaluronate eye drops (SH), the tear film break-up time was not significantly improved, while the corneal damage was significantly improved. Among the different dose pHDL treatment groups, the tear film break-up time of the mice in the medium dose group (10 mg / mL) and the high dose group (20 mg / mL) was significantly prolonged after treatment, and the corneal damage was significantly improved; the corneal damage of the mice in the low dose group (5 mg / mL) was also significantly improved after treatment.

[0038] 2.2.1 Hema-toxylin and eosin (HE) staining of cornea and lacrimal gland in each group of mice

[0039] 2.2.1 Hema-toxylin and eosin (HE) staining of cornea and lacrimal gland in each group of mice Figure 8 The normal group of mice showed normal corneal tissue structure, regular corneal epithelial cell morphology, clear layering, clear outline, uniform staining, and tight cell-cell connection. The epithelial cells were arranged in an orderly manner in about 3-4 layers, and the surface was smooth without thickening. The collagen fiber plate layer parallel to the cornea formed the stroma layer, and the scattered fibroblasts were distributed therein with tight arrangement. No inflammatory cell infiltration was observed in the tissue. Compared with the normal group, the BAC group showed obvious thickening of the corneal epithelial layer, increased cell layers, and a non-smooth corneal surface as indicated by the red arrow. The corneal epithelial cells showed partial edema with light staining of the cytoplasm. The collagen fibers in the stroma layer were arranged loosely, and the cell-cell connection was slightly loose as indicated by the blue arrow. Compared with the model group, the SHD group showed no improvement in the thickening of the corneal epithelial layer, and the cell membrane boundary was unclear and arranged in disorder. The collagen fibers in the stroma layer were arranged loosely. The pHDL 5 mg group showed a slightly thinner corneal epithelial layer, a smoother surface, but with epithelial cell shedding. The collagen fibers in the stroma layer were arranged in an orderly manner. The pHDL 10 mg group showed recovery of the corneal epithelial layer, a thicker corneal epithelial layer, a smooth surface, and an orderly arrangement of the stroma layer. The pHDL 20 mg group showed significant recovery of the cornea, regular and clear morphology of the epithelial layer cells, and tight arrangement of the stroma layer.

[0040] In addition, for the lacrimal gland, the normal group of mice showed lacrimal gland tissue composed of lobules of varying sizes, uniform size and regular morphology of acinar, tight arrangement of acinar cells, intact lacrimal gland epithelial cell structure, and high columnar shape with abundant secretory vacuoles in the cytoplasm. The BAC model group showed atrophy of the lacrimal gland cells, irregular arrangement, structural damage, reduced mucus in the lumen, uneven size and disordered arrangement of the acinar and lacrimal gland epithelium, and obvious swelling of the connective tissue with inflammatory cell infiltration in the interstitium. Compared with the model group, the SHD group showed reduced damage to the lacrimal gland tissue, slightly irregular arrangement of the acinar, and no obvious abnormalities. The pHDL 5 mg group showed severe damage to the lacrimal gland tissue, similar to the model group. The pHDL 10 mg group showed reduced pathological damage to the lacrimal gland tissue, with intact lacrimal gland structure and regular morphology of the acinar epithelial cells. The pHDL 20 mg group showed significant improvement, with relatively complete lacrimal gland structure.

[0041] 2.2.2 Periodic Acid-Schiff stain (PAS) staining for improving dry eye Figure 9 The conjunctival epithelial goblet cells of each group of mice were purple red in PAS staining and were round or oval. The number of goblet cells in the normal group was large, the shape was full, and the distribution was uniform. Compared with the normal group, the number of goblet cells in the BAC model group was reduced, the distribution was sparse, and part of the area was completely lost and the cell shape was small, as indicated by the purple arrow (goblet cell loss). Compared with the model group, the goblet cells in the SH group were slightly recovered, but there were still many missing; the pHDL dose groups were improved, and the number of goblet cells was different, among which the pHDL 20 mg group had more cells, and the shape was more full and uniform.

[0042] 2.3 Safety (ALT, AST, TP, ALB, LDH, CK, BUN, and CR) Figure 10 Serum of mice was collected, and ALT: glutamic-pyruvic transaminase; AST: glutamic-oxaloacetic transaminase; TP: total protein; ALB: albumin; LDH: lactate dehydrogenase; CK: creatine kinase; BUN: serum urea nitrogen; and CR: serum creatinine were detected. After benzalkonium chloride modeling, 0.1% sodium hyaluronate eye drops (SH), and pHDL intervention, serum biochemical detection was performed on mice, and no damage to the liver and kidney of mice was found. ALT: glutamic-pyruvic transaminase; AST: glutamic-oxaloacetic transaminase; TP: total protein; ALB: albumin; LDH: lactate dehydrogenase; CK: creatine kinase; BUN: serum urea nitrogen; and CR: serum creatinine.

[0043] The above only describes the preferred embodiments of the present application and is not intended to limit the present application in any form. Although the present application has been disclosed as above with the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make some changes or modifications to the above-mentioned technical content without departing from the technical solution of the present application, and any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application are still within the scope of the present application.

Claims

1. Use of a 1.4F dimer polypeptide complex with DMPC, characterized in that, The complex is used for preparing a medicine for treating dry eye syndrome. The gene sequence of the 4F dimer polypeptide is shown as SEQ ID NO:

1. The molar ratio of the 4F dimer polypeptide to DMPC is 1:50-1:

100. The particle size of the complex of the 4F dimer polypeptide and DMPC is 30 nm. The preparation method of the complex of the 4F dimer polypeptide and DMPC is as follows: (1) preparing an aqueous phase: dissolving the 4F dimer polypeptide in sterile water, mixing well, and reserving; (2) preparing an organic phase solution: dissolving DMPC in anhydrous ethanol, mixing well, and reserving; synthesizing the complex of the 4F dimer polypeptide and DMPC according to the flow rate of the organic phase 1 mL / min and the flow rate of the aqueous phase 5 mL / min, and after synthesis, the complex nanoparticles are concentrated by centrifugation at 4000 ×g for 15 minutes at 25°C using a 10 kD ultrafiltration tube, and then ddH2O is added and centrifuged for three times under the same centrifugation condition to elute the residual ethanol; The complex of the 4F dimer polypeptide and DMPC is prepared using physiological saline, the concentration of the 4F dimer polypeptide is 5-20 mg / ml, and the concentration of DMPC is 5-20 mg / ml. The dry eye syndrome is a water deficiency type, an evaporation overstrong type, or a mixed type.

2. Use according to claim 1, characterized in that, The complex of the 4F dimer polypeptide and DMPC further comprises a pharmaceutically acceptable carrier when used for preparing a medicine for treating dry eye syndrome.

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