Use of a composition containing N16 peptide and a transmembrane carrier in the preparation of a medicament for treating dry eye

Abstract

The present invention discloses the application of 55PIK inhibitors in the preparation of medicaments for treating dry eye.

Description

[Technical field]

[0001] The present invention relates to the field of biopharmaceuticals, more specifically to the application of P55PIK inhibitors in the preparation of medicaments for treating dry eye. [Background technology]

[0002] Dry eye is a disease that presents ocular symptoms and visual dysfunction due to tear film instability and / or damage to the ocular surface caused by abnormalities in tear quantity, quality or hydrodynamics. In China, various names that have emerged in clinical practice (such as dry eye, dry eye disease and dry eye syndrome) are collectively called "dry eye". The main contributing factors include old age, female gender, high altitude, diabetes, pterygium, air pollution, overuse of eye drops, use of video terminals, corneal refractive surgery, allergic eye diseases and some systemic diseases, which cause symptoms such as dry eyes, burning sensation, foreign body sensation, stinging sensation, itchy eyes, photophobia, red eyes, blurred vision and visual acuity fluctuation.

[0003] As the etiology of dry eye is complex, there is no unified classification standard for dry eye internationally, and there are various classification methods. In China, dry eye is classified according to the structure and function of the tear film on the ocular surface as follows: (1) Water deficiency type dry eye: Caused by insufficient aqueous tear production and / or abnormal quality. For example, dry eye caused by Sjogren's syndrome and many systemic factors. (2) Hyperevaporative type dry eye: Caused by abnormal quality or quantity of lipid layer. For example, meibomian gland dysfunction, blepharitis, video terminal syndrome, increased evaporation due to eyelid deficiency or abnormality, etc. (3) Mucin deficiency type dry eye: Caused by epithelial cell damage on the ocular surface. For example, damage caused by drug toxicity, chemical injury and burn to the ocular surface, and corneal limbal dysfunction. (4) Abnormal tear dynamics dry eye: Caused by abnormal tear dynamics. For example, abnormal blinking, delayed tear drainage and conjunctival relaxation. (5) Mixed dry eye: This is the most common type of dry eye seen in clinical practice and is caused by two or more of the above factors.

[0004] Dry eye is a disease linked to multiple factors of tears and ocular surface, which can damage the ocular surface, causing symptoms such as unstable tear film, eye discomfort, visual impairment, etc. Meanwhile, dry eye has complex etiology and pathological mechanism, long course, recurring disease, difficult to cure, and brings great inconvenience to patients in daily life and work, and seriously harms visual function.

[0005] Conventionally, dry eye has been treated with artificial tears, lubricating pastes, immunosuppressants, etc., and severe dry eye has been treated with serum or surgery. Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above-mentioned deficiencies or improvement needs of the prior art, the present invention provides the application of a 55PIK inhibitor in the preparation of a drug for treating dry eye, with the aim of providing a drug for treating dry eye that is easy to use and has a reliable therapeutic effect, thereby solving the problem that conventional drugs for treating dry eye only relieve or suppress symptoms and are not easy to prepare. [Means for solving the problem]

[0007] In order to achieve the above object, according to one aspect of the present invention, there is provided a method for preparing a medicament for treating dry eye comprising administering to said patient an N16 peptide having an amino acid sequence represented by SEQ ID NO: 1 and a transmembrane binding protein. Body and The present invention provides the use of a composition comprising:

[0008] Preferably, Use of the composition in which the transmembrane carrier is a transmembrane peptide or a liposome to provide.

[0009] Preferably, Use of a composition comprising a transmembrane peptide, the transmembrane peptide being linked to an N16 peptide by a peptide bond. to provide.

[0010] Preferably, Use of a composition, characterized in that the transmembrane peptide is a TAT peptide and the composition is a composition containing a TAT-N16 polypeptide having the amino acid sequence of SEQ ID NO:2. to provide.

[0011] Preferably, Use of the composition for the preparation of a medicament for treating moderate to severe hyperevaporative dry eye or dry eye with abnormal tear dynamics to provide.

[0012] According to another aspect of the present invention, a N16 peptide having the amino acid sequence SEQ ID NO: 1 and a transmembrane binding protein are Body and The present invention provides a drug for treating dry eye, which is a composition comprising:

[0013] Preferably, Drug for treating dry eye, in which the transmembrane carrier is a transmembrane peptide or a liposome to provide.

[0014] Preferably, the composition comprises the transmembrane peptide, and the transmembrane peptide is linked to the N16 peptide by a peptide bond. The present invention provides a combination drug for treating dry eye.

[0015] Preferably, The transmembrane peptide is a TAT peptide, and the composition contains a TAT-N16 polypeptide having an amino acid sequence of SEQ ID NO:2. To provide a drug for treating dry eye.

[0016] Preferably, The content of TAT-N16 polypeptide is at least 0.01% by weight. To provide a drug for treating dry eye. Effect of the Invention

[0017] Overall, compared with the prior art, the above solution according to the present invention can obtain the following beneficial effects:

[0018] Drugs containing the P55PIK inhibitor polypeptide of the present invention have good therapeutic effects on animal models of dry eye with little toxicity and side effects. At the same time, drugs containing p55PIK inhibitors also have significant therapeutic effects on dry eye patients. Therefore, p55PIK inhibitors have good prospects for development and are innovative drugs that can effectively treat dry eye, are safe, have low toxicity and have controllable quality. [Brief description of the drawings]

[0019] [Figure 1] Example 1: Detection results of protein p55PIK expression in normal control group and model group of rats with dry eye caused by tear hyposecretion [Diagram 2] Statistical chart of p55PIK gene detection results in normal control group and model group of rats suffering from dry eye caused by tear secretion deficiency (Example 1) [Diagram 3]Staining of the cornea of ​​dry eye with sodium fluorescein when treating corneal epithelial damage in rats with 0.1% TAT-N16 polypeptide (Example 1) [Figure 4] Goblet cell density in treated mice on day 7 in Example 2 [Diagram 5] Goblet cell density in treated New Zealand rabbits on day 7 of case 3; [Figure 6] Photographic record of case 4 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In order to make the objects, solutions and advantages of the present invention more clearly understandable, the present invention will be described in more detail below in conjunction with examples. Needless to say, the specific examples described herein are merely for the purpose of explaining the present invention, and do not limit the present invention. In addition, the technical features related to each implementation method of the present invention below can be combined with each other if they are not mutually inconsistent.

[0021] The drug for treating dry eye according to the present invention contains a P55PIK inhibitor, and preferably the content is at least 0.01% by weight.

[0022] Commonly used P55PIK inhibitors are polypeptide-based preparations, which have difficulty penetrating biological membranes. For example, N16 peptide has a serial number SEQ NO.1. In order to be suitable for topical ophthalmic formulations (such as eye drops), the above-mentioned dry eye treatment drugs need to have the ability to penetrate biological membranes, which can assist P55PIK inhibitors to penetrate biological membranes and improve their biological activity.

[0023] A transmembrane carrier can be employed, or an artificial transmembrane peptide can be connected to a polypeptide capable of inhibiting P55PIK so that it has the ability to penetrate biological membranes. For example, an N16 peptide can be connected to a TAT serial to form a TAT-N16 peptide. An example of the serial is SEQ NO.2.

[0024] Membrane penetrating peptide systems are divided into three types based on the serial of the membrane penetrating peptide and the binding properties of the membrane penetrating peptide with lipids: (1) Amphipathic penetrating systems. They usually contain more than 20 amino acids, with hydrophobic and hydrophilic amino acid residues arranged consecutively along the main structure. For example, transportan or TP10. (2) Intermediate amphipathic penetrating systems. They have fewer amino acids than amphipathic penetrating systems, and due to their amphipathicity, they form α-helixes or β-sheet structures when interacting with membranes. For example, penetratin, pVEC, and M918. (3) Non-amphipathic penetrating systems. They are usually short and contain a high proportion of positively charged amino acids (such as arginine). For example, R9 and TAT.

[0025] Transmembrane carriers, for example liposome encapsulation.

[0026] The researchers first applied TAT-N16 polypeptide (see SEQ.NO.2 for serial number) to animal models of dry eye. It was found that TAT-N16 polypeptide can prolong the break-up time of tear film and repair corneal damage, especially in cases of moderate to severe hyperevaporative dry eye and dry eye with abnormal tear dynamics. Further experiments showed that a transmembrane formulation (transmembrane carrier) containing P55PIK inhibitors was Body When the medicament for treating dry eye is used, a similar inhibitory effect can be observed, and it can be applied to the preparation of a medicament for treating dry eye.

[0027] Research has shown that the expression level of the protein p55PIK is abnormally increased in animal models of dry eye. Because the protein p55PIK participates in many important signaling pathways, inhibiting the activity of p55PIK can alleviate the symptoms of dry eye. Further experiments have shown that P55PIK inhibitors can repair corneal damage and prolong the break-up time of the tear film. EXAMPLES

[0028] p55PIK is expressed at increased protein levels in ocular tissues of animals with dry eye, and TAT-N16 polypeptide can alleviate the symptoms of dry eye in experimental rats.

[0029] 1. Materials and Methods 1.1 Animals, Reagents and Drugs Forty-two healthy SPF rats, half male and half female, weighing 150-300g, were selected, and 0.25% chloramphenicol eye drops were applied to both eyes 5 times a day at 0.05ml per eye. All animals were examined for abnormalities using a slit lamp before the start of the experiment. Reagents: 0.4% obvacaine hydrochloride eye drops, 0.5% lidocaine hydrochloride injection, 0.25% chloramphenicol eye drops, 1% benzalkonium bromide solution, 0.5% sodium carboxymethylcellulose eye drops, 0.2% carbomer eye drops, 0.9% NaCl injection, 0.5mg / ml atropine injection, 2% sodium fluorescein solution, and Schirmer measurement filter paper.

[0030] Slit lamp microscope, 5ml sterile syringe, No. 5 needle, tweezers, needle holder, ophthalmic scissors, stopwatch, suture needle, No. 1 black silk thread, antibody P55PIK (CST), GAPDH (ABclonal), TRizol reagent case, real-time quantitative PCRmIX, etc.

[0031] 1.2 Modeling and grouping 1.2.1 Rat corneal epithelium damage dry eye model: The lacrimal gland was removed and then benzalkonium bromide solution was applied to prepare a rat corneal epithelium damage dry eye model. 18 rats, half male and half female, were randomly selected, the hair around both eyes was removed, disinfected, topical anesthesia was administered with 0.4% obvacaine hydrochloride, and retrobulbar anesthesia was administered with 1ml of 0.5% lidocaine hydrochloride. A small hole was made in the skin in front of the rat's ear, and both lacrimal glands were peeled and removed. Both eyes were then applied with 1% benzalkonium bromide solution, and the corneal condition was examined the next day.

[0032] Grouping and treatment: 18 pre-formed rats, 6 per group, half male and half female, were randomly divided into 3 groups. Group 1 was a blank control group, untreated; Group 2 was a solvent control group, treated with a solvent containing no TAT-N16 polypeptide; Group 3 was a drug treatment group, treated with 0.1% TAT-N16 polypeptide eye drops. Eye drops were administered 4 times / (eye / day), 1 drop / time, for 7 days. The damage status of the corneal epithelium was recorded on day 0, 4 and 7 after treatment.

[0033] Detection method and scoring criteria: Sodium fluorescein corneal staining was performed by injecting one drop of sodium fluorescein into the conjunctival sac using a 5ml syringe and a No. 5 needle, and then examining the condition of the corneal epithelium 5 minutes later under a slit lamp microscope and cobalt blue light (see Table 1 for the scoring criteria).

[0034] 1.2.2 Rat model of lacrimal hyposecretion: An animal model of dry eye caused by lacrimal hyposecretion was created by referring to the Burgalassi method of injecting atropine into the lacrimal gland. 18 rats, half male and half female, were given 0.4% obvacaine hydrochloride eye drops in both eyes, and 0.25ml / eye (0.5mg / ml) of atropine was injected into the lacrimal gland under both eyes 4 times a day. The results of the rat lacrimal secretion test were recorded 0.5h after each atropine injection, and the injections were continued until the 7th day.

[0035] Grouping and treatment: Six rats, half male and half female, from the non-model group were divided into a normal control group. Eighteen model rats were randomly divided into three groups, six rats per group, half male and half female. The first model group was a blank control group, with no treatment; the second solvent control group was treated with a solvent that did not contain TAT-N16 polypeptide; the third drug treatment group was treated with 0.1% TAT-N16 polypeptide. Ophthalmic solution administration: 4 times / (eye / day), 1 drop / time, administered for 7 days. The tear secretion volume of each eye was recorded on day 0 after administration and on days 4 and 7 after administration. After detection was completed, the ocular detection p55PIK gene and protein levels of the normal control group and model group were taken.

[0036] 1.2.2.1 Tear secretion test (Schirmer II): The eyeball of SD rats is small, and it is not appropriate to use a filter paper of a size suitable for humans for measurement, so the test was performed by clamping it between both halves, and a 5×35mm filter paper was clamped between two 2.5×35mm filter papers, folded at the tip, and simply placed at the junction of the middle and back 1 / 3 of the lower eyelid, and after 5 minutes, the filter paper was removed and the wet length of the filter paper was measured three times every 2 minutes, and the average value was taken. The observation results before administration and on the 4th and 7th days were recorded.

[0037] 1.2.2.2 Western Blot Detection 1. Sample Preparation (1) After properly treating the eyeballs, they were washed 2-3 times with PBS and then placed in a denaturing lysis buffer containing 50-100 ul of protease inhibitors. (2) The sample was broken down by ultrasonic waves to collect the proteins. The EP pipe was inserted into the ice and placed in an ultrasonic breaker. The program was set to ultrasonic 5 s, pause 10, ultrasonic output 45%, and 5 cycles. (3) The mixture was centrifuged at 12,000 rpm at 4° C. for 10 minutes to remove cell fragments and foreign matter, and the supernatant was collected. (4) The supernatant was collected and the protein concentration was measured using Nanodrop and recorded. (5) 25 ul of sample buffer was added to the protein supernatant in a ratio of 5x, mixed uniformly, and placed in a metal bath for 10 minutes to denature the protein, thereby preparing the sample. Preparation of 5x protein supernatant sample buffer: TIFF0007641397000001.tif63114

[0038] 2. SDS-PAGE gel electrophoresis Preparation of SDS-PAGE gel. Calculate the volume according to the appropriately measured protein concentration, and add 10-20μg of protein to each sample supply hole. Electrophoresis voltage was set to constant voltage, first 80V, then switched to 120V after the sample entered the separation gel, and stopped electrophoresis after BPB overflowed from the gel.

[0039] 3. Transferring the PVDF Membrane in a Wet State Preparation of transfer buffer. After electrophoresis was completed, the gel was carefully peeled off, the concentrating gel was cut, and placed in the transfer buffer. The clips were aligned according to the polarity, and care was taken to ensure that there was no offset in the transfer due to the offset between each mezzanine in the clip. The electrophoresis transfer tank was placed in an ice box to lower the temperature, and transfer was performed at a constant current of 200 mA for 1.5 to 2 h.

[0040] 4. Immunocolorization The PVDF membrane transferred in a wet state was closed with skim milk powder. Antibody 1 was hybridized at room temperature for 2h or at 4℃ by the next morning. Antibody 1 was hybridized and then washed, and antibody 2 was hybridized and then hybridized at room temperature for 1h by shaking the culture bed. Be careful to avoid light during the operation of fluorescein antibody 2. Wash the membrane with antibody 2. Prepare the Odyssey two-color infrared laser imager until 20min before, adjust the laser intensity, and scan the protein signal according to the antibody 2 division.

[0041] 1.2.2.3 Gene discovery by real-time quantitative PCR detection of fluorescein 1. Eyeball Total RNA Extraction Total RNA was extracted according to the instructions in the TRizol reagent case. The extracted total RNA was measured by ultraviolet spectrophotometer, and the OD260 / 280 ratio of the extract concentration was in the range of 1.87 to 1.98. QPCR p55PIK primer serial: p55PIK-F: 5'-GCTTGGCACTTGATGTA -3' (SEQ NO. 3), p55PIK-R: 5'-GCTGTATGAAGAAGAATATAC-3' (SEQ NO. 4), primer serial of reference gene GAPDH: GAPDH-F: 5'-GGACCAGGTTGTCTCCTGTG-3' (SEQ NO. 5), GAPDH-R: 5'-TGTAGGCCATGAGGTCCAC-3' (SEQ NO. 6).

[0042] 2. Reverse transfer Reverse transfer was performed using TransScript® One-step gDNA Removal and cDNA Synthesis SuperMix (Beijing Quanshi Jin Co., Ltd.) according to the following procedure. 1) After mixing thoroughly, place in the CFX96 qPCR device and perform the amplification reaction under the following conditions. TIFF0007641397000002.tif461692) The following program was used for the dissolution curve. TIFF0007641397000003.tif37170Data analysis: Ct method was followed.

[0043] 2 Results 2.1 Rat corneal epithelial damage dry eye model Before treatment, the difference in corneal epithelial damage score between the groups was not statistically significant (P>0.05); after 4 days of treatment, the difference in corneal epithelial damage score between the drug group and the blank control group and the solvent control group was not statistically significant (P>0.05); after 7 days of treatment, the difference in corneal damage score between the drug group and the blank control group and the solvent control group was statistically significant (P<0.05). Among them, the corneal damage score of the drug group was significantly lower than that of the blank control group and the solvent control group (P<0.05) (Table 2). The comparison of sodium fluorescein staining and corneal condition is shown in Figure 3.

[0044] 2.2 Rat dry eye model with reduced tear secretion Before the modeling, the rats' tear secretion volume was (8.98±1.36) mm, but after the modeling, it decreased to (3.21±1.24) mm (P<0.001), indicating the success of the modeling. There was no statistically significant difference in tear secretion volume between the groups before and after the modeling (P>0.05), indicating that the tear secretion volume of each group was comparable. On the 4th and 7th days of treatment, the drug-treated group had significantly higher tear secretion volume than the control group (P<0.05) (Table 3).

[0045] Western blotting was used to detect the expression of protein p55PIK in ocular tissue samples from normal and model rats suffering from dry eye caused by decreased tear secretion. Expression of protein p55PIK in the model group eyeballs was significantly higher than that in the normal control group (Figure 1).

[0046] The transfer level of p55PIK gene in the eyes of rats with dry eye caused by tear hyposecretion was studied. RNA was extracted, reverse transferred, and real-time quantitative PCR was performed to detect the mRNA level. The study showed that the transfer level of p55PIK gene in the eyes of the model group was significantly higher than that in the normal control group (Figure 2). [Table 1] [Table 2] [Table 3]

[0047] Example 2: Mouse experiment in which TAT-N16 polypeptide relieves dry eye symptoms 1. Materials and Methods 1.1 Main materials and reagents 50 healthy male C57BL / 6 mice weighing 18-20g were used. They were healthy and had no abnormalities on the ocular surface. Reagents: 0.1% TAT-N16 peptide eye drops, 0.2% benzalkonium chloride solution, 1% sodium fluorescein solution, pentobarbital, phenol red cotton thread, PAS stain.

[0048] 2. Experimental process 2.1 Construction of a mouse dry eye model The C57BL / 6 mice were kept in a simulated environment of temperature (25±1)℃, relative humidity (25±5)%, and day-night shift (9AM-9PM). A 0.2% benzalkonium chloride solution was instilled into the eyes of 40 mice (80 eyes) twice a day (8AM, 6PM), 5μl per instillation, for 14 days to induce a dry eye model. The remaining 10 mice (20 eyes) were not treated and served as baseline controls.

[0049] 2.2 Evaluation of Dry Eye Models and Selection of Dosage On the 15th day after modeling, a dry eye model was selected according to tear film break-up time (BUT), corneal sodium fluorescein staining and tear secretion test (Schirmer I).

[0050] Tear film breakup time (BUT): The stability of the tear film was evaluated by the tear film breakup time commonly used in clinical practice. 1μl of 1% sodium fluorescein solution was applied to the mouse conjunctival sac, and an assistant held the mouse and artificially made it blink three times. The mouse was gradually held in a state where the upper and lower eyelids were separated, and immediately observed under a slit lamp microscope and cobalt blue light. The tear film breakup time at each time was the period when the first tear film rupture black spot appeared on the cornea. Measurements were repeated three times for each eye, and the average value was recorded (seconds).

[0051] Corneal fluorescein sodium staining: Corneal fluorescein sodium staining was used to determine whether the epithelium was damaged. One drop of 1% sodium fluorescein solution was placed in the conjunctival sac of the mouse, and the state of sodium fluorescein staining of the corneal epithelium was examined 1 minute later under a slit lamp microscope and cobalt blue light.

[0052] Scoring criteria: the cornea was divided into 4 quadrants and each quadrant was given a score of 0 to 4 for a total score of 16. Score 0: no staining, score 1: punctate staining in 30 or fewer spots, score 2: staining in 30 or more spots but not diffuse, score 3: marked diffuse staining but no patchy staining, and score 4: patchy staining.

[0053] Tear secretion test (Schirmer I): At the same time (3 PM) for each time point, Schirmer I tear secretion measurement was performed using phenol red cotton thread. After general anesthesia was administered to the mice by injecting pentobarbital (50 mg / kg body weight) into the abdominal cavity, the lower eyelid was lightly opened, and the folded end of the phenol red cotton thread, 1 mm long, was placed on the conjunctival surface of the eyelid close to the outer adjacent part of the lower eyelid for 15 seconds, and the length of the reddened part of the cotton thread was measured and recorded. After the measurement was completed, the eyelid crack was closed to prevent overexposure. The test was performed twice for each eye, and the average value was taken as the final score.

[0054] Conjunctival goblet cell count: Changes in the number of conjunctival goblet cells were examined by cutting paraffin slices from the conjunctival fundus and staining with PAS. The blank and solvent groups had significantly fewer conjunctival goblet cells than normal conjunctiva. After treatment with TAT-N16 polypeptide eye drops, the number of conjunctival goblet cells increased, and the difference was statistically significant.

[0055] 2.3 Grouping and treatment of dry eye model Thirty mice (60 eyes) of the model of dry eye that met the above criteria were selected and randomly divided into groups A, B, and C, with half of each sex per group. Among them, 0.1% TAT-N16 polypeptide eye drops were administered to group A four times a day, eye drops not containing TAT-N16 polypeptide (solvent administration group) were administered to group B four times a day, and group C (control group) was not treated. On the 1st, 4th, and 7th days after administration, BUT, Schirmer I, and sodium fluorescein staining were performed, respectively, and on the 7th day, paraffin slices from the conjunctival fundus were stained with PAS, and the number of positively stained goblet cells was counted.

[0056] 3. Grades: After establishing the dry eye mouse model, the treatment effect of TAT-N16 polypeptide ophthalmic solution on dry eye was examined by administering the solution to each mouse for 7 consecutive days. On the 4th day, the tear film breakup time was longer in the TAT-N16 polypeptide ophthalmic solution group than in the blank control group, model control group, and solvent group, the tear secretion amount was significantly increased in the TAT-N16 polypeptide ophthalmic solution group than in the blank control group and solvent group, and the corneal fluorescein sodium staining score was lower in the TAT-N16 polypeptide ophthalmic solution group than in the blank control group, model control group, and solvent group. After 7 consecutive days of eye drops, the tear film breakup time was significantly longer in the TAT-N16 polypeptide eye drop group than in the blank control group, model control group, and solvent control group, the difference being statistically significant (P=0.007) (Table 4), and the tear secretion volume was significantly increased in the TAT-N16 polypeptide eye drop group than in the blank control group and solvent control group, the difference being statistically significant (P<0.05) (Table 5). The corneal fluorescein sodium staining score was significantly lower in the TAT-N16 polypeptide eye drop group than in the blank control group, model control group, and solvent control group, the difference being statistically significant (P<0.05) (Table 6). In addition, the conjunctival goblet cell density in the TAT-N16 polypeptide eye drop group was significantly higher than in the blank control group and solvent control group, the difference being statistically significant (P<0.01) (Figure 4). [Table 4] [Table 5] [Table 6]

[0057] Example 3: TAT-N16 peptide relieves dry eye symptoms (Experiment with New Zealand rabbits) 1. Materials and Methods 1.1 Material 1.1.1 The experimental animals were 30 healthy New Zealand rabbits, both male and female, weighing between 2.1 and 2.5 kg. Animal care and surroundings complied with international standards for animal experiments in ophthalmic and vision scientific research.

[0058] 1.1.2 Main reagents: 0.1% TAT-N16 polypeptide eye drops, benzalkonium chloride (BAC) (Sigma) prepared in deionized water to a concentration of 0.1%, test filter paper for tear secretion test (Schirmer), sodium fluorescein ophthalmic detection test paper, nitrocellulose membrane (PALL), periodic acid, Schiff (PAS) staining reagent case.

[0059] 1.2 Method 1.2.1 Grouping of experimental animals: Thirty New Zealand rabbits were randomly assigned into blank control group, solvent control group and drug treatment groups, with 10 rabbits per group, according to the numerical table method.

[0060] 1.2.2 Creation of animal models The right eye of each animal was used as the experimental eye, and 0.1% benzalkonium chloride was administered twice a day for 4 weeks to create an animal model of dry eye. After 4 weeks, the rabbit model of dry eye was examined. The cornea and conjunctiva were dry in appearance, the amount of tears was small, the tear secretion test (Schirmer) test value was 10mm / 5min or less (10mm or more in the control group), the tear film breakup time (BUT) was 10s or less, and cornea and conjunctival dots and flat, light staining were visible in fluorescein staining. The model was deemed to have been successfully created.

[0061] 1.2.3 Preparation and use of medicines 0.1% TAT-N16 polypeptide eye drops is a liquid preparation, the raw materials of which include polypeptide raw materials and physiological saline. Specific preparation method: Take 0.1% of the polypeptide raw materials by weight and dissolve them in physiological saline. Instill 0.1% TAT-N16 polypeptide eye drops three times a day, 2 drops each time, for 7 days. The blank control group received no treatment, and the solvent control group was instilled with physiological saline three times a day, 2 drops each time, for 7 days, with the normal eyes serving as the normal control group.

[0062] 1.3 Sampling and detection Tests on experimental animals should be performed by the same person. Tests should be performed at the same time, place, and under the same lighting conditions. Before administration and on the 4th and 7th days after administration, the rabbits' corneal and conjunctival appearance, fluorescein staining, and tear film breakup time should be examined with a slit lamp. At the same time, tear secretion time tests and conjunctival blot cytology tests (CIC) should be performed.

[0063] 1.3.1 Tear secretion test (Schirmer) After administering topical anesthesia with obvacaine hydrochloride and waiting 1 minute, liquid was applied around the eyelids of the dry eye with a cotton swab, and the test paper was placed at the junction of the middle and outer third of the rabbit's inferior conjunctival sac. The remainder was suspended on the surface of the skin and the eye was closed. After 5 minutes, the filter paper was removed and the length of the wet filter paper was measured.

[0064] 1.3.2 Tear film breakup time experiment A sodium fluorescein ophthalmic detection test strip is placed in the conjunctival sac of the lower eyelid for a short period of time, and the sodium fluorescein is allowed to be distributed evenly over the surface of the eyeball. Then, with the eyelids open, observation is performed using a slit lamp microscope and cobalt blue light until the first dry spots appear on the tear film. The period from opening of the eyelids to the appearance of the first dry spots is recorded, and measurements are taken three times in a row, with the average value being recorded.

[0065] 1.3.3 Conjunctival Blot Cytology (CIC) On the 1st, 4th, and 7th days after administration, conjunctival blot cytology was performed for each group. Nitrocellulose membranes were sandwiched into 3.5 x 3.5 mm pieces, soaked in distilled water for 4 hours, and then dried. After topical anesthesia, two pieces of nitrocellulose membrane were placed on the bulbar conjunctiva of the upper quadrant of the nose and temple with the rough side facing down, and pressed continuously for 10 seconds to remove the superficial epithelial cells. The filter membranes were fixed in 95% alcohol and stained with periodic acid-Schiff's reagent (PAS). Goblet cells were counted under a light microscope and graded according to the Nelson scoring standard.

[0066] 2 Results 2.1 Schirmer test The rabbit dry eye model value was less than 10 mm, and the Schirmer test values ​​before administration in the blank control group, the solvent control group and the drug-treated group were (8.41±0.69) mm, (8.50±0.23) mm and (8.38±0.57) mm, respectively, with no significant difference. On the fourth day of treatment, the Schirmer test value of the drug-treated group was (12.02±1.21) mm, which was significantly improved from before administration and significantly improved compared with the blank control group and the solvent control group. After 7 days of administration, the Schirmer test value of the drug-treated group was (16.31±1.45) mm, which was significantly extended compared with before administration and significantly extended compared with the blank control group and the solvent control group (see Table 7).

[0067] 2.2 BUT The breakup time of the tear film was less than 10s. Before administration, the BUT values ​​of the blank control group, the solvent control group and the drug-treated group were (8.55±1.06) s, (8.11±0.82) s and (8.24±1.13) s, respectively, which showed no significant difference. On the fourth day of treatment, the drug-treated group had a BUT value of (11.72±1.27) s, which was significantly longer than before administration and significantly longer than the blank control group and the solvent control group; after 7 days of administration, the drug-treated group had a BUT value of (15.31±1.85) s, which was significantly longer than before administration and significantly longer than the blank control group and the solvent control group (see Table 8).

[0068] 2.3 Sodium fluorescein staining (FL) After the dry eye model was created, the rabbits' corneas were observed with the naked eye to be dry and not shiny, and fluorescein staining revealed corneal and conjunctival dots and flat, light staining. After 4 days of administration to the drug-treated group, the corneas of the rabbits were less dry than before administration, the amount of tears increased, and the corneas and flat, light staining were reduced, and after 7 days of treatment, the corneas and conjunctivae were wet, the amount of tears increased significantly, and no staining was observed.

[0069] 2.4 Conjunctival Blot Cytology (CIC) The difference in goblet cell density between the dry eye model group and the normal control group was statistically significant. After administration, the goblet cell density in the drug-treated group gradually increased, and the difference from before administration was statistically significant (see Figure 5). [Table 7] [Table 8]

[0070] Example 4: Human experimentation In order to further prove the therapeutic effect of the eye drops, a 0.1% TAT-N16 polypeptide eye drop according to the present invention is being administered to volunteers for a test purpose.

[0071] Case 1: Ms. Wu, female, 70 years old, had been suffering from dry eye, itchy eyes, astringent eyes, foreign body sensation and photophobia for 3 years. She was diagnosed with dry eye. She has always used artificial tears, but it only relieves the symptoms in a short time and the duration of action is short. 0.1% polypeptide eye drops were administered, 1-2 drops per time, 3 times a day. On the first day, the symptoms of dry eye were relieved, and the effect was greater than that of artificial tears. On the second day, the eyes felt wet, the dry eye was relieved, and the irritation symptoms such as photophobia and astringent eyes were significantly relieved. On the third day, the eyes felt wet, and the symptoms of dry eye, itchy eyes and foreign body sensation were significantly relieved, and the therapeutic effect was fast, and the eyes felt comfortable when pointed to 3 points. The effect of the medicine lasted for a long time, and after one week of administration, the symptoms of dry eye were almost completely gone. The photographic record is shown in Figure 6 (1).

[0072] Case 2: Ms. Huang, female, 63 years old, had symptoms of dry eye, itchy eyes, and tearing when she felt a cold for more than a year. She had a little conjunctival congestion and was diagnosed with dry eye. She had been administered artificial tears and levofloxacin eye drops from time to time, but the treatment was not effective and the symptoms recurred. She was administered 0.1% polypeptide eye drops, 1-2 drops per time, 3 times a day. On the first day, the symptoms of dry eye and itchy eyes were significantly relieved, and the ocular comfort was enhanced. On the second day, the conjunctival congestion disappeared, and the eyes were wet. On the fourth day, the symptoms of tearing when she felt a cold were significantly reduced, and the eyes felt comfortable. After one week of continuous administration, the symptoms of conjunctival congestion disappeared, and the symptoms of dry eye were almost completely gone. The photographic record is shown in Figure 6 (2).

[0073] Case 3: Mr. Xu, male, 35 years old, IT worker, had a medical history of dry eye for more than one year and was diagnosed with dry eye. Although the symptoms were relieved with artificial tears in a short time, the dry eye recurred and the biconjunctiva was congested. 0.1% polypeptide eye drops were administered, 1-2 drops three times a day. About 5 minutes after administration, the eyes felt comfortable and wet, and the effect of the drug was sustained for a long time. After administration on the third day, the symptoms of dry eye were significantly relieved, and after continuous administration for one week, the symptoms of dry eye were almost completely gone. There was no feedback that dry eye recurred after stopping the administration. According to the patient, the effect was remarkable and the effect of the drug was sustained for a long time. The photographic record is shown in Figure 6 (3).

[0074] Case 4: Ms. Kyung, female, 67 years old, dry eye, foreign body sensation, photophobia, 2 years history, slight congestion of eyelids and conjunctiva, diagnosed as dry eye, used eye drops (classification unknown), but the effect was undesirable, and glasses were required when outdoors. 0.1% polypeptide eye drops were administered, 3-5 times a day, 1-2 drops each time. 5 minutes after applying, she felt comfortable, and photophobia was reduced. After 1 day, conjunctival congestion was reduced, and she did not need to wear glasses outdoors for a certain period of time. After continuous administration for 1 week, eyelid conjunctival congestion disappeared, dry eye and photophobia symptoms were almost completely gone, and she rarely wears glasses outdoors, except when she feels uncomfortable in a special place. Photo record is shown in Figure 6 (4).

[0075] As can be easily understood by those skilled in the art, the above are merely preferred examples of the present invention, and are not intended to limit the present invention. All modifications, equivalent changes and improvements that comply with the spirit and principles of the present invention are subject to the patent protection of the present invention.

Claims

1. 2. Use of a composition comprising an N16 peptide having an amino acid sequence of SEQ ID NO: 1 and a transmembrane carrier, the composition being used for the preparation of a medicament for treating dry eye.

2. 2. Use of the composition according to claim 1, characterized in that the transmembrane carrier is a transmembrane peptide or a liposome.

3. 3. The use of the composition according to claim 2, characterized in that the composition comprises the membrane-spanning peptide, and the membrane-spanning peptide is linked to the N16 peptide by a peptide bond.

4. The membrane-spanning peptide is a TAT peptide, 4. Use of the composition according to claim 3, characterized in that the composition is a composition containing a TAT-N16 polypeptide whose amino acid sequence is SEQ ID NO:

2.

5. 2. Use of the composition according to claim 1, characterized in that it is used for the preparation of a medicine for treating moderate to severe dry eye of hyperevaporative type or dry eye of abnormal tear dynamics.

6. A drug for treating dry eye, characterized in that it is a composition containing an N16 peptide whose amino acid sequence is SEQ ID NO: 1 and a transmembrane carrier.

7. The drug for treating dry eye according to claim 6, wherein the transmembrane carrier is a transmembrane peptide or a liposome.

8. The drug for treating dry eye according to claim 7, characterized in that the composition comprises the membrane-spanning peptide, and the membrane-spanning peptide is linked to the N16 peptide by a peptide bond.

9. The membrane-spanning peptide is a TAT peptide, 9. The drug for treating dry eye according to claim 8, wherein the composition contains a TAT-N16 polypeptide having an amino acid sequence represented by SEQ ID NO:

2.

10. The drug for treating dry eye according to claim 9, characterized in that the content of the TAT-N16 polypeptide is at least 0.01% by weight.

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