Polypeptide for improving bioavailability of medicine and application thereof

Abstract

The invention provides a polypeptide for improving the bioavailability of a medicine and application of the polypeptide, and belongs to the technical field of biological medicines. The length of the polypeptide sequence is 3-30 amino acids, the number of cysteine accounts for 10%-50% of the total number of amino acids of the polypeptide, and 1-6 amino acids are arranged between every two adjacent cysteines. The polypeptide provided by the invention has no influence on drug activity, is high in biological safety, and does not cause eye tissue damage. The polypeptide provided by the invention can be widely compatible in ophthalmic drugs as a drug partner, improves the residence time of the drugs in an eye drop preparation on the ocular surface, and improves the ability of the drugs to permeate the eye barrier, thereby improving the bioavailability of the drugs in the eye drop preparation, and improving the bioavailability of the drugs in the eye drop preparation. The eye drops have very important application prospects in the field of treating various eye diseases through eye drop administration.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a polypeptide for improving the bioavailability of a drug and an application thereof. Background Art

[0002] The eye is one of the most sophisticated and complex organs in the human body. In order to ensure the normal function of the eye, the eye has produced a series of biological barriers in the long-term evolution to prevent the invasion and damage of foreign substances, but it also prevents the effective delivery of drugs in the eye. These biological barriers mainly include: corneal barrier, blood-retinal barrier (BRB), physiological barrier and other barriers. The cornea is divided into epithelial cell layer, anterior elastic layer, stroma layer, posterior elastic layer and endothelial cell layer. Based on the dual barrier effect of the corneal epithelial cell layer and stroma layer, only a few drug molecules with suitable oil-water partition coefficient and dissociation constant (pKa) have good corneal permeability. The retina is a soft and transparent membrane that is closely attached to the inner surface of the choroid. There are tight junctions between the top cells of the retinal capillary endothelium, which hinder the penetration of molecules with a diameter of 2nm and above. In addition, physiological barriers (such as tear circulation, aqueous humor circulation and blood-aqueous humor barrier) and other barriers (such as drug efflux, transport proteins, and ocular metabolic enzymes) will sharply reduce the concentration of drugs reaching the eye. Due to the existence of the above-mentioned biological barriers, there are many challenges in the delivery of ocular drugs.

[0003] Compared with invasive drug delivery, eye drops are a safe and convenient drug delivery method in ophthalmology, with high patient acceptance. However, eye drops need to overcome more biological barriers, which makes the bioavailability of drugs in eye drops extremely low. It is reported that for small molecule drugs, when eye drops are administered topically, the proportion of drugs reaching the front of the eye is between 3% and 5%, and the proportion of drugs reaching the back of the eye is almost zero. For biological macromolecules such as antibody drugs, growth factor drugs, and nucleic acid drugs that have weaker ability to penetrate tissue barriers, it is almost impossible to achieve therapeutic drug concentrations in the eye through eye drops.

[0004] The use of penetration enhancers can improve the bioavailability of drug molecules in eye drops. At present, benzalkonium chloride, cell-penetrating peptides, calcium ion chelators, surfactants, etc. have been used as penetration enhancers in eye drops to improve the bioavailability of drugs in eye drops. The long-term use of the above-mentioned penetration enhancers has obvious side effects. For example, benzalkonium chloride, as a commonly used preservative and surfactant, may cause eye irritation, conjunctivitis, corneal damage, decreased vision, allergic reactions, and may even induce dry eyes. In addition, calcium ion chelators may interfere with the normal physiological functions of cells, resulting in decreased stability of the corneal epithelium and increased risk of eye infections. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a polypeptide for improving the bioavailability of drugs, which can break through many barriers in the eye, promote the endocytosis of drug molecules by cells, and improve the bioavailability of drugs in eye drops.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A polypeptide for improving the bioavailability of drugs, wherein the polypeptide sequence length is 3 to 30 amino acids, the number of cysteine ​​is 10% to 50% of the total number of amino acids in the polypeptide, and the interval between two adjacent cysteines is 1 to 6 amino acids.

[0008] Preferably, the amino acid sequence of the polypeptide is selected from any one of the following: SEQ ID NO.1: CRRC RRCRR; SEQ ID NO.2: CHRCHRCHR; SEQ ID NO.3: CRYCRYCRY; SEQ ID NO.4: CRICRICRI; SEQ ID NO.5: IRCIRCIRC; SEQ ID NO.6: CRHCYE; SEQ ID NO.7: HCIECYRCRCRR; SEQ ID NO.8: RCYCERRECHHID; SEQ ID NO.9: HCYHCIDIYECRCRRRCH; SEQ ID NO.10: GCRGCYGGCRRHCYERCRR; SEQ ID NO.11: CYRGHCHRC; SEQ ID NO.12: CYRCHRCGECRRYCRCR CI; SEQ ID NO.13: CYRCIGCRGCYGGCRRHCYERCR; SEQ ID NO.14: CHRGCYR CIGCRGCYGGCRRHCYERCRRCH; SEQ ID NO.15: IDIYECRCYRCHRC GECRRYCRCRCICH.

[0009] Another object of the present invention is to provide a preparation for improving the bioavailability of a drug, wherein the preparation comprises the polypeptide or a pharmaceutically acceptable salt thereof.

[0010] Another object of the present invention is to provide the use of the polypeptide or its pharmaceutically acceptable salt or the preparation in the preparation of drugs for treating eye diseases.

[0011] Preferably, the mass ratio of the active pharmaceutical molecule in the drug to the polypeptide is 1:(0.001-10).

[0012] Preferably, the polypeptide can open the eye barrier and promote the diffusion of drugs into the eye.

[0013] Preferably, the polypeptide can prolong the retention time of the drug on the ocular surface.

[0014] Preferably, the polypeptide improves the efficiency of drug absorption by ocular cells.

[0015] Preferably, the eye diseases include anterior eye diseases and posterior eye diseases.

[0016] Preferably, the anterior eye diseases include dry eye, corneal neovascularization, corneal scarless repair and keratitis; the posterior eye diseases include retinal neovascularization, choroidal neovascularization, macular degeneration, glaucoma and optic nerve repair.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a polypeptide for improving the bioavailability of a drug. The polypeptide provided by the present invention that can be used as a drug chaperone is a class of cysteine-rich polypeptides, which can break through many eye barriers, promote the endocytosis of drug molecules by cells, enhance the bioavailability of drugs in eye drops, and improve the therapeutic effect of drugs in eye diseases; the compatibility mode with drug molecules is simple and does not change the biological activity of drug molecules; the polypeptide provided by the present invention has high safety and will not cause damage to eye tissues when used for a long time.

[0019] After the polypeptide provided by the present invention carries the drug, it can be retained on the ocular surface for a long time when instilled on the ocular surface, increasing the probability of the drug passing through the ocular barrier. Not only can it be effectively taken up by corneal epithelial cells, it can deliver the drug to the target tissue through a transcellular pathway, and it also has the function of efficiently opening the tight junctions between corneal epithelial cells, passing through the corneal barrier through a paracellular pathway, and delivering the drug to the eye. It realizes the delivery of various ophthalmic drugs in the form of non-invasive eye drops, improves their bioavailability, and improves the efficacy of the original drug on ocular diseases, and is expected to be widely used in the treatment of ocular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a comparison chart of the activities of different drugs in Example 1;

[0021] Figure 2 The fluorescence microscope images of green fluorescent protein expressed by different drugs in ocular surface epithelial cells in Example 2;

[0022] Figure 3 Fluorescence microscope images of different drugs in Example 3 after co-incubation with ocular surface epithelial cells for 2 hours;

[0023] Figure 4 The expression levels of inflammatory factors (TNF-a and IL-6) of different drugs in the inflammatory epithelial cell model stimulated by phospholipid polysaccharide (LPS) in Example 4; ****p<0.0001;

[0024] Figure 5 The graphs are (a) a comparison of the retention time of epidermal growth factor in different drugs on the ocular surface of rats and (b) a graph of the mean fluorescence intensity in Example 5;

[0025] Figure 6 This is a fluorescence microscope picture of the transcytosed endocytosis of the drug in the ocular surface epithelial cells in Example 6;

[0026] Figure 7 These are light microscope images of different drugs passing through the multilayer corneal epithelial cell model in Example 7;

[0027] Figure 8 The results of staining for tight junction protein occludin expression before and after polypeptide treatment in Example 8; a is a fluorescence microscope image, the upper row is without polypeptide treatment, and the lower row is after polypeptide treatment; b is the average fluorescence intensity statistics (expression amount); ****p<0.0001;

[0028] Fig. 9 The distribution diagram of bevacizumab drug concentration in various parts of the eye tissue at different time points after different drugs were applied to the eye in Example 9; wherein a represents free bevacizumab after eye application; b represents bevacizumab + polypeptide after eye application;

[0029] Fig.10 The effects of different drugs in Example 10 in treating the rat alkali burn corneal neovascularization model; a is a representative slit lamp photo of the front of the rat eyeball; b is the percentage statistics of the neovascularization area to the total corneal area; c is the statistical analysis of inflammatory indicators; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significant difference;

[0030] Fig.11 The effects of different drugs in Example 11 in treating the dry eye model in rats; wherein a is the tear outflow; b is the statistics of corneal sensitivity; ***p<0.001, ns, no significant difference;

[0031] Fig.12 The effects of different drugs on the oxygen-induced retinal neovascularization model in Example 12; a is a representative retinal flat mount IB4 staining image, IB4 (red); b is the statistical analysis result of the ratio of the area of ​​non-vascular perfusion area to the area of ​​the retina; c is the statistical analysis result of the ratio of the area of ​​pathological blood vessels to the area of ​​the retina; ***p < 0.001, ns, no significant difference;

[0032] Fig.13 The statistics of intraocular pressure of different drugs in Example 13 in the treatment of rat glaucoma model; ***p<0.001, ns, no significant difference;

[0033] Fig.14 The morphology and number of corneal endothelial cells after 60 days of eye drops with the polypeptide of Example 14 are shown in Figure 14; wherein a is a slit lamp examination result of the anterior segment of the eye of a rat; b is a trypan blue-alizarin red staining of corneal endothelial cells of a rat; c is a statistical result of the number of corneal endothelial cells of a rat; ns is not significantly different;

[0034] Fig.15 The structure and inflammatory infiltration of the retina 60 days after the polypeptide instillation of Example 15; wherein, a is the a / b wave amplitude of the electroretinogram (ERG), and b is the H&E staining picture of the retina; ns, the difference is not significant. DETAILED DESCRIPTION

[0035] The invention provides a polypeptide for improving the bioavailability of drugs. The polypeptide sequence length is 3 to 30 amino acids, the number of cysteine ​​is 10% to 50% of the total number of amino acids in the polypeptide, and 1 to 6 amino acids are spaced between two adjacent cysteines.

[0036] In the present invention, the amino acid sequence of the polypeptide is selected from any one of the following: SEQ ID NO.1: CRRCRR CRR; SEQ ID NO.2: CHRCHRCHR; SEQ ID NO.3: CRYCRYCRY; SEQ ID NO.4: CRICRICRI; SEQ ID NO.5: IRCIRCIRC; SEQ ID NO.6: C RHCYE; SEQ ID NO.7: HCIECYRCRCRR; SEQ ID NO.8: RCYCERRECHHID; SEQ ID NO.9: HCYHCIDIYECRCRRRCH; SEQ ID NO.10: GCRGCYGGCRRHCYERCRR; SEQ ID NO.11: CYRGHCHRC; SEQ ID NO.12: CYRCHRCGECRR YCRCRCI; SEQ ID NO.13: CYRCIGCRGCYGGCRRHCYERCR; SEQ ID NO.14: CHRGCYRCIGCRGCYGGCRRHCYERCRRCH; SEQ ID NO.15: IDIYECRCYR CHRCGECRRYCRCRCICH.

[0037] The present invention also provides a preparation for improving the bioavailability of a drug, wherein the preparation comprises the polypeptide or a pharmaceutically acceptable salt thereof.

[0038] The polypeptide provided by the present invention can effectively improve the bioavailability of active drug molecules; the drugs include water-soluble drugs and fat-soluble drugs; the active drug molecules include macromolecular drugs and small molecule drugs; the macromolecular drugs include antibody drugs, growth factor drugs, nucleic acid drugs, and polypeptide drugs, the molecular weight of the antibody drugs and growth factor drugs is greater than 2kDa, the number of base units of nucleic acid drugs is greater than 20bp, and the number of polypeptide drugs is greater than 15 amino acid sequences; the small molecule drugs include antibiotics, hormones, and non-steroidal drugs, and their molecular weight is not greater than 5kDa.

[0039] The present invention also provides the use of the polypeptide or its pharmaceutically acceptable salt or the preparation in preparing a drug for treating eye diseases. As an practicable manner, the drug for treating eye diseases of the present invention is eye drops.

[0040] In the present invention, in the drug for treating eye diseases, the mass ratio of the active drug molecule in the drug to the polypeptide is 1:(0.001-10); the drug and the polypeptide are dissolved separately and then mixed to obtain an eye drug with high bioavailability, and the polypeptide of the present invention does not change the processing technology of the original drug preparation, nor does it affect the activity of the original drug. The drug and the polypeptide in the present invention can be made of conventional solvents in the art.

[0041] In the present invention, the polypeptide can open the eye barrier and promote the diffusion of drugs into the eye; preferably, it can open the tight barrier of the cornea.

[0042] In the present invention, the polypeptide can prolong the retention time of the drug on the ocular surface. The polypeptide of the present invention can prolong the retention time of the drug on the ocular surface to more than 3 hours, and the drug retention rate within 3 hours is greater than 40%. The present invention improves the bioavailability of the drug by prolonging the retention time of the drug on the ocular surface.

[0043] In the present invention, the polypeptide improves the absorption efficiency of drugs in eye cells. The polypeptide of the present invention can increase the speed and ability of eye cells to internalize drugs, enhance the transcellular endocytosis ability, and thus effectively increase the absorption efficiency of drugs.

[0044] In the present invention, the eye diseases include anterior eye diseases and posterior eye diseases; the anterior eye diseases include dry eye, corneal neovascularization, corneal scarless repair and keratitis; the posterior eye diseases include retinal neovascularization, choroidal neovascularization, macular degeneration, glaucoma and optic nerve repair.

[0045] The polypeptide of the present invention can effectively increase the drug concentration of drugs (macromolecule drugs or small molecule drugs) in drugs for treating eye diseases in the anterior tissues of the eye (cornea, conjunctiva and aqueous humor) and the posterior tissues of the eye (retina, choroid, sclera and vitreous body).

[0046] The polypeptide of the present invention has high biological safety. The drug for treating eye diseases prepared from the polypeptide of the present invention is administered three times a day with an interval of 8 hours between each administration. After continuous administration for 60 days, no damage is caused to the eye tissue. The integrity of the cornea on the eye surface is good, the structure of the retinal endothelial cells is intact, the function of the eye tissue is normal, and no inflammation or adverse reactions occur.

[0047] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] Example 1

[0049] ① Antibody drug bevacizumab 1 mg / mL (referred to as free bevacizumab), ② 1 mg / mL bevacizumab + 1 mg / mL peptide (CRRCRRCRR) (referred to as bevacizumab + drug partner peptide) were dissolved in phosphate buffer (0.01M, pH 7.4), placed at 4°C for 24 hours, and then bevacizumab activity test kit (purchased from Shanghai Ruifan Biotechnology Co., Ltd.) was used to test the activity of bevacizumab. The test method was carried out according to the kit manufacturer. The activity test results are shown in Figure 1 As shown, the drug chaperone peptide did not affect the activity of bevacizumab.

[0050] Example 2

[0051] In a 24-well plate, a glass slide was built into the well plate, and ocular surface epithelial cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were cultured at a cell density of 1×10 5 After 24 hours of cell attachment, the ocular surface epithelial cells were divided into two groups, and ①1 mL of cell culture medium containing 500 μg of green fluorescent protein particles (gene drugs) (referred to as green fluorescent protein particles), and ②1 mL of cell culture medium containing 500 μg of green fluorescent protein particles + 0.5 mg of polypeptide (CHRCHRCHR) (referred to as green fluorescent protein particles + drug chaperone peptide) were added respectively. After 24 hours of co-incubation, the slides were removed, washed three times with phosphate buffer (concentration 0.01M, pH 7.4), fixed with 4% paraformaldehyde for 15 minutes, and washed three times with PBS; permeabilized with 0.5% TritonX-100 for 10 minutes, and washed three times with PBS; nonspecific antibodies were blocked with 5% BSA for 30 minutes, and the cells were washed three times with PBS, stained with DAPI (5μg / mL) for 20 minutes, and washed three times with PBS on a decolorizing shaker, and anti-fluorescence quenching agent was added to the adhesive glass slide, the cell slide was removed, and the side with the adherent cells was inverted on the glass slide, and the slide was sealed with nail polish. The expression of fluorescent proteins in cells was observed under a laser confocal microscope. Figure 2As shown, in sample ①, no expression of green fluorescent protein was observed in the cells, while in sample ②, the expression of green fluorescent protein in the cells was clearly observed. The results show that the polypeptide of the present invention effectively promotes the diffusion of drugs into cells.

[0052] Example 3

[0053] In a 24-well plate, a glass slide was built into the well plate, and ocular surface epithelial cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were cultured at a cell density of 1×10 5 Each well was planted, and after 24 hours of cell attachment, the ocular surface epithelial cells were divided into two groups, and ①1 mL of cell culture medium containing 1 mg of fluorescently labeled bevacizumab (protein drug) (recorded as fluorescently labeled free bevacizumab) and ②1 mL of cell culture medium containing 1 mg of fluorescently labeled bevacizumab + 0.1 mg of peptide (CRYCRYCRY) (recorded as fluorescently labeled free bevacizumab + drug partner peptide) were added respectively. After 24 hours of co-incubation, the slides were taken out, washed 3 times with phosphate buffer, fixed with 4% paraformaldehyde for 15 minutes, and washed 3 times with PBS; 0.5% TritonX-100 was permeabilized for 10 minutes, and the cells were washed 3 times with PBS, and non-specific antibodies were blocked with 5% BSA for 30 minutes, and the cells were washed 3 times with PBS; DAPI (5 μg / mL) was stained for 20 minutes, and the cells were washed 3 times with PBS on a decolorizing shaker, and anti-fluorescence quenching agent was added to the adhesive slide, and the cell slide was taken out and the side of the adherent cells was turned upside down on the slide, and the slide was sealed with nail polish. The penetration of proteins through the cell membrane was observed under a laser confocal microscope. Figure 3 As shown, in sample ①, no green fluorescent protein was observed to penetrate the cell membrane, while in sample ②, green fluorescent protein was clearly observed to penetrate the cell membrane. The results show that the polypeptide of the present invention effectively promotes the diffusion of drugs into cells.

[0054] Example 4

[0055] In a 24-well plate, a glass slide was built into the well plate, and ocular surface epithelial cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were cultured at a cell density of 1×10 5100 cells / well were planted therein, and after 24 hours of cell attachment, lipopolysaccharide (LPS; 1 mg / mL) was used to induce the cells to express inflammatory cytokines. After 24 hours of induction, RAW 264.7 cells treated with LPS (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were used as controls. The ocular surface epithelial cells were divided into three groups, namely the model group (denoted as phospholipid polysaccharide), and the addition of ①1 mL of cell culture medium containing 1 mg of dexamethasone (hormone drug) (denoted as phospholipid polysaccharide + dexamethasone), and ②1 mL of cell culture medium containing 1 mg of dexamethasone + 0.05 mg of polypeptide (CRICRICRI) (denoted as phospholipid polysaccharide + dexamethasone + drug chaperone peptide). Total RNA was extracted using the Novozyme RNA extraction kit. Complementary DNA (cDNA) templates were synthesized by reverse transcription according to the manufacturer's instructions. RT-qPCR was performed using a Roche light cycler480 fluorescence quantitative PCR instrument (Roche). 2 -ΔΔCt Calculate the relative expression levels of inflammatory factors (TNF-a and IL-6). Figure 4 As shown, compared with the cells in the inflammation model, the levels of TNF-a and IL-6 secreted by cells in ① and ② were decreased; compared with the levels of TNF-a and IL-6 secreted by cells in ①, the levels of TNF-a and IL-6 secreted by cells in ② were even lower, indicating that the drug chaperone peptide increased the utilization of dexamethasone by cells.

[0056] Example 5

[0057] Ten normal SD rats were randomly divided into two groups and given drug ① free fluorescently labeled epidermal growth factor (growth factor drug) and drug ② free fluorescently labeled epidermal growth factor + epidermal growth factor in peptide (IRCIRCIRC) samples. After the two groups of rats were anesthetized with isoflurane gas, ① 20 μL of drug containing (concentration 1 mg / mL) free fluorescently labeled epidermal growth factor (referred to as fluorescently labeled raw epidermal growth factor) and ② 20 μL of drug containing (concentration 1 mg / mL) free fluorescently labeled epidermal growth factor + peptide (IRCIRCIRC) (referred to as fluorescently labeled raw epidermal growth factor + drug partner peptide) were dripped into the rats' eyeballs. Then, at preset time points (0, 0.5, 1, 2, 3 and 4 hours), the head area of ​​the rats was imaged and photographed using a multimodal in vivo imaging system. Fluorescence quantitative data were analyzed using the software that comes with the instrument. Figure 5 As shown, after 1 hour, there was basically no fluorescently labeled epidermal growth factor retained on the surface of group ①, and after 4 hours, 50% of the fluorescently labeled epidermal growth factor was still retained on the surface of group ②, and within 3 hours, the total amount of fluorescently labeled epidermal growth factor retained on the surface exceeded 60%. The results show that drug chaperone peptides can significantly enhance the retention time of drug chaperone peptide epidermal growth factor on the ocular surface.

[0058] Example 6

[0059] HCECs cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were divided into 5×10 4 The cells were inoculated on the cell slides in the 24-well plate at a density of 100 cells / mL and placed in a cell culture incubator for 48 hours to a suitable density, and then divided into two experimental groups: group i and group ii. Group ii was not treated with any treatment, and the cells in group i were treated with 1 mg / mL fluorescently labeled bevacizumab + peptide (CRHCYE) for 3 hours, and then 1 mg / mL fluorescently labeled bevacizumab + drug partner peptide CRHCYE was removed and washed with PBS for 3 times; the slides of group i and group ii were placed in the same well of the 6-well plate and incubated for another 3 hours. Both groups were subjected to immunofluorescence staining. At the end of the incubation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, and washed three times with PBS; permeabilized with 0.5% TritonX-100 for 10 minutes, and washed three times with PBS; nonspecific antibodies were blocked with 5% BSA for 30 minutes, and washed three times with PBS; DAPI (5μg / mL) was stained for 20 minutes, and the cells were washed three times with PBS on a decolorizing shaker. Anti-fluorescence quenching agent was added to the adhesive slide, and the cell slide was taken out and the side of the adherent cells was inverted on the slide, and the slide was sealed with nail polish. The laser confocal microscope was used to take pictures and record. Figure 6 As shown, fluorescently labeled bevacizumab was observed to enter cells in both group i and group ii, indicating that the drug chaperone peptide CRHCYE has the effect of assisting bevacizumab transcytosis.

[0060] Example 7

[0061] The reconstructed three-dimensional corneal model (source: Guangzhou Boxi Biotech Co., Ltd.) in the Transwell plate was transferred to a sterile 12-well cell plate for culture. ①300 μL of BioGrowth cell culture medium containing 200 μg / mL fluorescently labeled epidermal growth factor (growth factor drug) (referred to as fluorescently labeled raw epidermal growth factor) and ②300 μL of BioGrowth cell culture medium containing 200 μg / mL fluorescently labeled epidermal growth factor + peptide (HCIECYRCRCRR) (referred to as fluorescently labeled epidermal growth factor + drug partner peptide) were added to the inside of the Transwell plate. The control group was added with BioGrowth cell culture medium. A total of 700 μL BioGrowth cell culture medium was added to the outside of the Transwell plate, which was then placed in a cell culture incubator for 24 hours. The plate was removed and rinsed with PBS. It was fixed with paraformaldehyde and stained with DAPI. A membrane with three-dimensional multilayered corneal epithelium was cut off at the bottom of the Transwell plate. It was placed on a glass slide and sealed with an antiquencher. Imaging was performed using a laser scanning confocal microscope. like Figure 7As shown, in ①, almost no fluorescently labeled epidermal growth factor can be seen passing through the recombinant three-dimensional corneal cell model, and ② fluorescently labeled epidermal growth factor is found to pass through the recombinant three-dimensional corneal cell model, indicating that the polypeptide HCIECYRCRCRR can assist the epidermal growth factor to pass through the recombinant three-dimensional corneal model.

[0062] Example 8

[0063] 5×10 per well 4 HCEC cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were inoculated into 24-well plates at a density of cells and cultured in an incubator for 5 days. After 5 days of culture, tight junction proteins were stably expressed between HCEC cells, and then the culture medium was discarded. A culture medium containing the polypeptide RCYCERRECHHID (at a concentration of 50 μg / mL) was added, and the cells were cultured for another 6 hours. After the culture technique, the cells were rinsed three times with sterile PBS. The cells were fixed with 4% paraformaldehyde, permeabilized with TritonX-100, and sealed with 5% BSA solution at room temperature. After incubation at room temperature for three hours, Occludin (human tight junction protein) antibody was added to the cell nucleus, and then DAPI was added for staining. Then an anti-fluorescence quencher was added, and then sealed with nail polish. The prepared samples were then observed and photographed using a confocal laser scanning microscope (CLSM). Figure 8 As shown in the upper row of a in FIG, the tight junction protein Occludin is highly intact between cells in the sample (denoted as a single-layer corneal epithelial cell model) that has not been treated with the peptide RCYCERRECHHID. Figure 8 As shown in the lower row of a in Figure 1, the tight junction protein Occludin in the sample treated with the peptide RCYCERRECHHID (denoted as the drug chaperone peptide) was significantly deteriorated between cells. The quantitative analysis results of the tight junction protein Occludin are shown in Figure 1. Figure 8 As shown in b, the expression of tight junction protein Occludin in the sample treated with peptide RCYCERRECHHID decreased significantly, indicating that peptide RCYCERRECHHID can open the tight junctions of corneal epithelial cells.

[0064] Example 9

[0065] The experiment was conducted using C57 / 6b mice, which were divided into two groups, with 5 mice in each group: ① 5 μL of 1 mg / mL free bevacizumab was dropped into the eye, and ② 5 μL of 1 mg / mL bevacizumab + 0.05 mg / mL peptide (HCYHCIDIYECRCRRRCH) was dropped into the eye. The mice were then killed at different time points (1, 3, 6, and 12 hours), the eyeballs were removed, and the eye tissues were separated. The tissues were then ground using an automatic sample freezing grinder (frequency 60 Hz, time 480 s), and then centrifuged at 13,000 rpm and 4°C for 5 minutes, and the supernatant was collected. The free bevacizumab ElisaKit was used to detect the drug concentration of free bevacizumab in the eye tissues at different time points. Fig. 9 As shown in a, bevacizumab was not detected in almost all ocular tissues in ①; Fig. 9 As shown in b, bevacizumab can be detected in various ocular tissues in ②, indicating that the peptide HCYHCIDIYECRCRRRCH can assist protein drugs to enter ocular tissues and increase the concentration of protein drugs in various ocular tissues.

[0066] Example 10

[0067] Establishment of rat corneal neovascularization model. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg / kg). After anesthesia, local anesthetics were used to dilate the pupil. A 3 mm diameter round filter paper disk (containing 3 μL 1 M sodium hydroxide solution) was placed in the center of the rat cornea for 40 seconds. The conjunctival sac was continuously rinsed with 20 mL precooled (4°C) 0.9% sodium chloride solution, and the residual liquid in the conjunctival sac was removed with a sterile cotton swab. The above neovascularization model mice were divided into 4 groups, 10 in each group: ① model group, given 20 μL normal saline, ② 20 μL 1 mg / mL dexamethasone (hormone drug) eye drop group, ③ 5 μL 4 mg / mL dexamethasone subconjunctival injection group, ④ 20 μL 1 mg / mL dexamethasone + 1 mg / mL peptide (GCRGCYGGCRRHCYERCRR) eye drop group, treatment of rat alkali burn corneal neovascularization, 3 times a day, on the 3rd, 7th and 14th day of treatment, the anterior segment of the rat was taken with a slit lamp. According to the inflammatory index grading standard (Table 1), the length of the new blood vessels was quantified, and its area was calculated as follows:

[0068]

[0069] The letter S represents the area of ​​new blood vessels, which is calculated by multiplying the number of circumferential hours of new blood vessels (represented by the letter c) by the corneal radius (represented by the letter r). The values ​​of r and L were calculated by Image J software, and the final data were expressed as the percentage of the calculated area to the total corneal area. According to the inflammatory index grading standard (Table 1), photos were taken and the anterior segment inflammatory index of rats in each group was evaluated on the 3rd, 7th, and 14th days after alkali burns. The final inflammatory index was the sum of the scores divided by 9, and then statistical analysis was performed using statistical software. Fig.10 As shown in a, the representative slit lamp photo of the front of the rat eyeball, on the third day, the corneas of the rats in each group showed varying degrees of edema, and no obvious new blood vessels were observed in any quadrant of the cornea. Fig.10 b In the statistics of the percentage of the neovascular area to the total corneal area, and Fig.10 The statistical analysis of the c inflammatory indexes found that compared with the model group, the order of the groups in reducing neovascularization and reducing inflammatory indexes was: ②<③<④. This result shows that the polypeptide GCRGCYGGCRRHCYERCRR has the ability to improve the effect of dexamethasone in treating ocular surface neovascularization.

[0070] Table 1 Grading standards of inflammation index

[0071]

[0072] Embodiment 11

[0073] Construction of a dry eye mouse model: 0.2% benzalkonium chloride was added to a PBS solution and topically applied to the eyes of BALB / c mice three times a day (8 a.m., 2:30 p.m., and 9 p.m.) for 10 days. Subsequently, these dry eye mouse models treated with benzalkonium chloride were randomly divided into the following 4 groups, with 16 to 18 mice in each group: ① normal mice, no medication, ② model group, given normal saline, ③ 0.1 mg / mL cyclosporine (peptide drug) eye drops, ④ 0.1 mg / mL cyclosporine + peptide (CYRGHCHRC) eye drops. The treatment method was 5 μL per eye three times a day (8:30 a.m., 3:00 p.m., and 9:30 p.m.) for 10 consecutive days. During the treatment period, eye drops were applied three times a day (8:00 a.m., 2:30 p.m., and 9:00 p.m.). At 4 p.m. on the 10th day, all mice were evaluated for tear outflow and corneal sensitivity tests. Fig.11 As shown, relative to the normal group, in the test of increasing (a) tear outflow and increasing (b) corneal sensitivity, the treatment effect of each group is in the following order: ②<③<④. This result indicates that the polypeptide CYRGHCHRC has the ability to improve the effect of cyclosporine in treating dry eye.

[0074] Example 12

[0075] Construction of oxygen-induced fundus neovascularization model: 7-day-old newborn mice and female mother mice were placed in an oxygen chamber with an oxygen content of 75%. During this period, the mother mice prepared in advance were replaced every 24 hours. After 5 days, the mice were returned to the normal environment, and the mice that met the modeling weight (greater than 5.0g) were randomly divided into 6 groups, with no less than 20 mice in each group: ① normal group, no medication, ② disease model group, given normal saline, ③ eye drops of bevacizumab, ④ eye drops of peptides, ⑤ intravitreal injection of bevacizumab, ⑥ eye drops of bevacizumab + peptides, to treat oxygen-induced retinal neovascularization model. The medication started on the 12th day, twice a day, 5μL of formula eye drops each time, the concentration of bevacizumab was 12.5mg / mL, the concentration of peptide (CYRCHRCGECRRYCRCRCI) was 1mg / mL, and the retinal neovascularization of mice was monitored on the 17th day, and the experimental data were statistically analyzed. ① Normal group, ② Disease model group, ③ Eye drops of bevacizumab, ④ Eye drops of peptide, ⑤ Intravitreal injection of bevacizumab, ⑥ Eye drops of drug chaperone peptide and (bevacizumab + drug chaperone peptide) were used to treat oxygen-induced retinal neovascularization model. At the selected time point (5 days after administration), the eyeballs were removed, placed in 4% PFA, and fixed at room temperature for about 1 hour. The anterior segment of the eyeball was removed, and the retina was separated from the choroid with microforceps. The retina was cut into four petals using microscissors and stored in PBS at 4°C. The retinal blood vessels were stained with Isolectin-GS B4 staining solution (0.5% Triton-X100 + 0.5% Isolectin-GS B4 + 1mmol / L CaCl2) at 4°C for 12 hours. After washing and sealing, images were captured using a laser confocal microscope. PS was used to analyze the area of ​​retinal non-perfusion and neovascularization. As shown Fig.12 As shown, relative to the normal group, according to (a) representative retinal flat mount IB4 staining images, (b) statistical analysis results of the ratio of the area of ​​non-vascular perfusion area to the area of ​​the retina, and (c) statistical analysis results of the ratio of the area of ​​pathological blood vessels to the area of ​​the retina, the treatment effect of each group is in the following order: ②≈③≈④<⑤<⑥. This result indicates that the peptide CYRCHRCGECRRYCR CRCI has the ability to improve the effect of bevacizumab in treating fundus neovascularization.

[0076] Example 13

[0077] Construction of glaucoma model: First, 0.1% betamethasone eye drops were used to induce elevated intraocular pressure, once a day for two weeks. A 5.5g and 10.0g double plunger load meter was used to measure intraocular pressure. Modeling was considered successful when the intraocular pressure reached or exceeded 22 mmHg. The rabbits were divided into 4 groups, with 8 rabbits in each group: ① normal group, no medication, ② model group, normal saline, ③ 50 μL 2 mg / mL brimonidine (non-somal drug) was applied to the eyes, and ④ 50 μL 2 mg / mL brimonidine + 1 mg / mL peptide (CYRCIGCRGCYGGCRRHCY ERCR) was applied to the eyes. Intraocular pressure was measured at different time points (0, 1, 2, 3, 4, 5, 6, 8, 10, 12 and 14 days). Fig.13 As shown, compared with the normal group, the order of the effects of each group in lowering intraocular pressure is: ②<③<④. The results indicate that the polypeptide CYRCIGCRGCYGGCRRHCYERCR can improve the therapeutic effect of brimonidine in the treatment of glaucoma.

[0078] Embodiment 14

[0079] Mice were eye-doped with 5 mg / mL polypeptide CHRGCYRCIGCRGCYGGCRRHCYERCRRCH for 60 days (referred to as eye-doping partner peptide), three times a day, 5 μL each time, with an interval of 8 hours. The anterior segment of the eyes of mice and rats was photographed using a slit lamp to observe corneal edema and anterior chamber depth, and sodium fluorescein was used to observe whether the cornea was stained or defective. H&E sections of the eyeballs were processed. The structure and inflammatory infiltration of the cornea were observed under an upright microscope. After SD rats were administered with the corresponding treatment formula, they were anesthetized and killed, and the cornea was immediately removed and cut into a four-leaf clover shape. Then, 0.25% trypan blue was used for staining for 2 minutes, and after rinsing 3 times with normal saline, 0.2% alizarin red with pH = 4.2 was used for staining for 2 minutes and rinsed 3 times. The morphology of corneal endothelial cells was observed under a wet mount microscope, and the number of hexagonal endothelial cells in the same field of view was counted and statistically analyzed. Fig.14 As shown in a, compared with the non-eye-drop group, the slit-lamp examination results of the anterior segment of the eye of the rats in the eye-drop group showed that the cornea was intact; Fig.14 As shown in b, trypan blue-alizarin red staining of rat corneal endothelial cells showed that the endothelial structure was intact; Fig.14 As shown in c, the statistical results of the number of rat corneal endothelial cells showed that the number of endothelial cells did not change significantly. This result shows that the polypeptide CHRGCYRCIGCRGCYGGCRRHCYERCRRCH has no effect on the structure of the cornea on the ocular surface and has good biocompatibility.

[0080] Embodiment 15

[0081] 5 mg / mL peptide IDIYECRCYRCHRCGECRRYCRCRCICH was used for 60 days (referred to as eye-drop chaperone peptide), three times a day, 5 μL each time, with an interval of 8 hours, and the mice were dark-adapted overnight. The whole experimental environment was kept dark. Chloramine (50 mg kg -1 ) and xylazine (5 mg kg -1 ) After anesthesia, tropicamide was topically dripped to dilate the pupil. After lidocaine topical anesthesia, the electrodes of Roland electrophysiology were correctly connected: the negative electrodes (blue) of the two channels were inserted into the subcutaneous part of the mouse's forehead using needle electrodes; the positive electrodes (red) of the two channels were connected to the corneas of both eyes using gold ring electrodes; the common ground electrode (black) was inserted into the subcutaneous part near the mouse's tail using a needle electrode. Finally, the light-adapted and dark-adapted 3.0ERG retinal electrical signals were detected, and the amplitudes of the a and b waves were statistically analyzed. H&E sections of the eyeballs were processed. Pictures were taken under an upright microscope to observe the structure of the retinal part and the inflammatory infiltration. As Fig.15 As shown in a, the a / b wave amplitude of the electroretinogram (ERG) in the eye-dot group did not change significantly compared with the non-eye-dot group. Fig.15 As shown in b, the H&E staining picture of the retina shows that the retinal structure is intact. This result shows that the peptide IDIYECRCYRCHRCGECRRYCRCRCICH has no effect on the functional structure of the cornea of ​​the fundus and has good biocompatibility.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide for improving the bioavailability of a drug, characterized in that: The length of the polypeptide sequence is 3 to 30 amino acids, the number of cysteine ​​is 10% to 50% of the total number of amino acids in the polypeptide, and the interval between two adjacent cysteines is 1 to 6 amino acids.

2. The polypeptide according to claim 1, characterized in that The amino acid sequence of the polypeptide is selected from any one of the following: SEQ ID NO.1: CRRCRRCRR; SEQ ID NO.2: CHRCHRCHR; SEQ ID NO.3: CRYCRYCRY; SEQ ID NO.4: CRICRICRI; SEQ ID NO.5: IRCIRCIRC; SEQ ID NO.6: CRHCYE; SEQ ID NO.7: HCIECYRCRCRR; SEQ ID NO.8:RCYCERRECHHID; SEQ ID NO.9: HCYHCIDIYECRCRRRCH; SEQ ID NO.10: GCRGCYGGCRRHCYERCRR; SEQ ID NO.11: CYRGHCHRC; SEQ ID NO.12: CYRCHRCGECRRYCRCRCI; SEQ ID NO.13: CYRCIGCRGCYGGCRRHCYERCR; SEQ ID NO.14: CHRGCYRCICGRGCYGGCRRHCYERCRRCH; SEQ ID NO. 15: IDIYECRCYRCHRCGECRRYCRCRCICH.

3. A preparation for improving the bioavailability of a drug, characterized in that: The preparation comprises the polypeptide according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.

4. Use of the polypeptide according to claim 1 or 2 or a pharmaceutically acceptable salt thereof or the preparation according to claim 3 in the preparation of a drug for treating eye diseases.

5. The use according to claim 4, characterized in that: The mass ratio of the active drug molecule in the drug to the polypeptide is 1:(0.001-10).

6. The use according to claim 4, characterized in that: The polypeptide can open the eye barrier and promote the diffusion of drugs into the eye.

7. The use according to claim 4, characterized in that: The polypeptide can prolong the retention time of the drug on the ocular surface.

8. The use according to claim 4, characterized in that: The polypeptide improves the drug absorption efficiency of eye cells.

9. The use according to claim 4, characterized in that: The eye diseases include anterior eye diseases and posterior eye diseases.

10. The use according to claim 9, characterized in that: The anterior eye diseases include dry eye, corneal neovascularization, corneal scarless repair and keratitis; the posterior eye diseases include retinal neovascularization, choroidal neovascularization, macular degeneration, glaucoma and optic nerve repair.

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