Novel artificial tear containing recombinant human lysozyme

Abstract

The invention provides a novel artificial tear containing recombinant human lysozyme, the novel artificial tear comprises a main component and an auxiliary material, the main component is recombinant human lysozyme, and based on the total mass of the novel artificial tear, the content of the recombinant human lysozyme is greater than or equal to 0.015% and less than 0.075%. The novel artificial tear contains recombinant human lysozyme with lower concentration and has better repairing and anti-inflammatory effects, and compared with the high-concentration artificial tear and the low-concentration artificial tear, the novel artificial tear has more obvious effects of increasing tear secretion, promoting cornea repairing and resisting inflammation. The raw material of the recombinant human lysozyme is derived from the recombinant human lysozyme stock solution, and the pH and osmotic pressure molar concentration value adjustment is not needed in the artificial tear preparation process, so that the large-scale production process can be greatly simplified, and the large-scale production stability is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a new type of artificial tears containing recombinant human lysozyme, belonging to the technical field of medicine. BACKGROUND

[0002] Dry eye syndrome refers to a variety of diseases caused by any reason, which leads to abnormal quality or dynamics of tear, and results in decreased stability of tear film, accompanied by eye discomfort and / or ocular surface tissue lesion characteristics. In recent years, with the increasing popularity of electronic products, and the high intensity of eye use of the population, the prevalence of dry eye syndrome such as eye fatigue and eye inflammation is increasing. According to statistics, in recent years, the number of patients with dry eye disease in cities is increasing rapidly by 10%-20% per year.

[0003] Human natural tears are a kind of transparent liquid secreted by human lacrimal glands and other organs, which is composed of inorganic salts, polysaccharides, proteins, lipids and other substances, and has multiple functions such as barrier, antibacterial, bactericidal and immune regulation, which plays an important role in protecting eyeball, nourishing ocular surface tissue and perfecting visual function.

[0004] At present, the artificial tears on the market for tear secretion deficiency type dry eye caused by various reasons are mainly based on basic liquid supplement and moisturizing, and most of them contain chemical bacteriostatic agents. Although these products can temporarily relieve dry eye discomfort, long-term contact with bacteriostatic agents may cause allergic, inflammatory and other irritation symptoms, and may also cause iatrogenic keratitis. Numerous experiments have proved that the bacteriostatic agent in eye drops has cytotoxicity, which can affect the function and normal metabolism of cells, and if used for a long time, it can cause damage to the ocular surface tissue and reduce the tolerance of the eye to eye drops. Some also form drug-induced conjunctivitis and drug-induced dry eye. Not only can't solve the problem of dry eye, but also may aggravate the inflammation of the eye tissue.

[0005] The artificial tears in ZL201810614434.9 need to add a step of adjusting pH, which has little effect on small-scale tests, but when large-scale (30 kg and above) production is carried out, pH adjustment will introduce a great disturbance factor, lengthen the process cycle and increase the process difficulty. SUMMARY

[0006] The present application provides a new type of artificial tears containing recombinant human lysozyme, and optimizes the large-scale production process. The new type of artificial tears can effectively relieve and treat dry eye syndrome, and has significant anti-inflammatory effect.

[0007] To achieve the above-mentioned purpose, the present application provides a new type of artificial tears containing recombinant human lysozyme, which comprises a main component and an auxiliary material, the main component is recombinant human lysozyme, and the content of recombinant human lysozyme is 0.015% to less than 0.075% based on the total mass of the new type of artificial tears.

[0008] According to a specific embodiment of the present application, preferably, the content of recombinant human lysozyme is 0.0375%≤content <0.075, based on the total mass of the new artificial tears.

[0009] According to a specific embodiment of the present application, preferably, the content of recombinant human lysozyme is 0.01875%-0.0375%, based on the total mass of the new artificial tears.

[0010] According to a specific embodiment of the present application, preferably, the amino acid sequence of the recombinant human lysozyme is completely consistent with that of human natural lysozyme. The recombinant human lysozyme used in the present application is obtained by microbial fermentation, and is 100% homologous to the amino acid sequence of natural lysozyme in the human body.

[0011] The recombinant human lysozyme (rhLYZ, see patent ZL201810614434.9, ZL 201710736505.8, ZL 201710233245.2) used in the present application can be obtained by microbial fermentation, preferably by fermentation and purification of Pichia pastoris engineering bacteria expressing human lysozyme, and has a molecular weight of 14700D. The amino acid sequence of the obtained recombinant human lysozyme is 100% identical to that of natural human lysozyme, and is homologous to the human body. The amino acid sequence is as follows: KVFERCELAR TLKRLGMDGY RGISLANWMC LAKWESGYNT RATNYNAGDR STDYGIFQINSRYWCNDGKT PGAVNACHLS CSALLQDNIA DAVACAKRVV RDPQGIRAWV AWRNRCQNRD VRQYVQGCGV (SEQ ID NO: 1).

[0012] According to a specific embodiment of the present application, preferably, the raw material of the recombinant human lysozyme is derived from a recombinant human lysozyme stock solution.

[0013] According to a specific embodiment of the present application, preferably, the recombinant human lysozyme stock solution is a colorless and clear liquid.

[0014] According to a specific embodiment of the present application, preferably, the content of sodium chloride in the recombinant human lysozyme stock solution is 0.36-0.44 w / v%.

[0015] According to a specific embodiment of the present application, preferably, the purity of recombinant human lysozyme in the recombinant human lysozyme stock solution is ≥97.0%.

[0016] According to a specific embodiment of the present application, preferably, the content of recombinant human lysozyme in the recombinant human lysozyme stock solution is ≥3mg / mL.

[0017] According to the specific embodiment of the present application, preferably, the recombinant human lysozyme stock solution does not contain disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0018] In the present application, the recombinant human lysozyme stock solution does not contain disodium hydrogen phosphate and sodium dihydrogen phosphate, i.e. the content of disodium hydrogen phosphate and sodium dihydrogen phosphate is 0. If disodium hydrogen phosphate and sodium dihydrogen phosphate exist in the recombinant human lysozyme stock solution, it will cause pH fluctuation in the artificial tear when the artificial tear is prepared.

[0019] According to the specific embodiment of the present application, preferably, the auxiliary material comprises a pH stabilizer.

[0020] According to the specific embodiment of the present application, preferably, the pH stabilizer is a buffer solution composed of disodium hydrogen phosphate and sodium dihydrogen phosphate; more preferably, the disodium hydrogen phosphate is anhydrous disodium hydrogen phosphate and the sodium dihydrogen phosphate is monohydrate sodium dihydrogen phosphate; further preferably, the mass ratio of the anhydrous disodium hydrogen phosphate to the monohydrate sodium dihydrogen phosphate is (2.6-7.0):(7.0-11.2), further preferably 5.3:8.6.

[0021] In the present application, the mass ratio of the anhydrous disodium hydrogen phosphate to the monohydrate sodium dihydrogen phosphate is within the range, and under this ratio, pH adjustment is not needed when the new artificial tear is prepared on a large scale. Other ratios will cause certain pH fluctuation when the new artificial tear is prepared on a large scale, so that the pH deviates from the set median value, and at this time, a pH regulator needs to be introduced for pH adjustment, which will make the preparation process of the new artificial tear more complicated.

[0022] According to the specific embodiment of the present application, preferably, 260 mOsmol / kg≤the osmotic pressure molar concentration of the new artificial tear <285 mOsmol / kg; more preferably, the osmotic pressure molar concentration of the new artificial tear is 270-280 mOsmol / kg.

[0023] The new artificial tear of the present application is slightly hypotonic, and the osmotic pressure molar concentration value of the ocular surface of the dry eye patient is relatively high, so that supplementing the new artificial tear of the present application which is slightly hypotonic is helpful to help the osmotic pressure molar concentration value of the ocular surface return to normal isotonic state.

[0024] The present application believes that the ideal artificial tear should have components, physicochemical properties and biological functions close to natural tears; the ocular surface is directly exposed to the external environment, and a healthy eye continuously secretes tears and coats to form a stable tear film through blinking movement, and the tear film is the only barrier for the ocular surface to isolate from the external environment. The biological function of natural tears is to bear the protection of the ocular surface, which is the comprehensive physiological function produced by the cooperation of each component of the tear.

[0025] The tear secretion of dry eye patients is reduced, the evaporation amount is large, and the natural tear is concentrated to become "high-osmotic tear". From the perspective of osmotic pressure, artificial tears should be isotonic solution, so the osmotic pressure of the prescription artificial tears should not be higher than that of natural tears, or slightly lower than that of natural tears, and be controlled to be slightly hypotonic.

[0026] According to the specific embodiments of the present application, preferably, the adjuvant comprises sodium hyaluronate and / or sodium chloride.

[0027] According to the specific embodiments of the present application, preferably, the content of sodium hyaluronate is 0.05-0.15%, more preferably 0.10%, based on the total mass of the new artificial tears.

[0028] According to the specific embodiments of the present application, preferably, the content of sodium chloride is 0.70%-0.80%, based on the total mass of the new artificial tears, and the content of sodium chloride is calculated as pure solid; more preferably, the content of sodium chloride in the new artificial tears is 0.80% (including 0.4% sodium chloride contained in the recombinant human prolysozyme liquid).

[0029] According to the specific embodiments of the present application, preferably, the pH value of the new artificial tears is 6.0-7.0, preferably 6.2-6.8, more preferably 6.4-6.6.

[0030] According to the specific embodiments of the present application, preferably, the new artificial tears comprise, based on 10000 parts by weight of the total amount of the new artificial tears: 1.5 parts ≤ the amount of added recombinant human lysozyme < 7.5 parts, 5.0-15.0 parts of sodium hyaluronate, 1.75-8.66 parts of anhydrous sodium hydrogen phosphate, 5.38-12.10 parts of sodium dihydrogen phosphate monohydrate, 70.0-80.0 parts of sodium chloride, and 9883.46-9909.65 parts of water.

[0031] In some specific embodiments of the present application, based on 10000 parts by weight of the total amount of the new artificial tears, the new artificial tears comprise: 4.0 parts of recombinant human lysozyme, 10.0 parts of sodium hyaluronate, 5.33 parts of anhydrous sodium hydrogen phosphate, 8.62 parts of sodium dihydrogen phosphate monohydrate, 80.0 parts of sodium chloride (including 0.4% sodium chloride contained in the recombinant human prolysozyme liquid), and 9892.05 parts of water.

[0032] According to the specific embodiments of the present application, preferably, based on 10000 parts by weight of the total amount of the new artificial tears, the new artificial tears comprise: 3.75 parts of recombinant human lysozyme, 10.0 parts of sodium hyaluronate, 5.33 parts of anhydrous sodium hydrogen phosphate, 8.62 parts of sodium dihydrogen phosphate monohydrate, 80.0 parts of sodium chloride (including 0.4% sodium chloride contained in the recombinant human prolysozyme liquid), and 9892.30 parts of water.

[0033] The preparation steps of the recombinant human lysozyme stock solution of the present application include:

[0034] The preparation steps are shown in Example 1 of ZL 201810614434.9; the difference is that, in the 3.2.3 section 0099 of Example 1 of ZL 201810614434.9, after the eluate is purified by Sephadex G25 gel chromatography, 0.4% sodium chloride is added as the mobile phase for buffer replacement to obtain a replacement solution, and the obtained replacement solution is subjected to 0.22 um filtration to remove bacteria to obtain the recombinant human lysozyme stock solution.

[0035] Compared with ZL 201810614434.9, the recombinant human lysozyme stock solution of the present application has the following technical effects when applied to human lysozyme artificial tears:

[0036] pH system stability: the change of osmotic pressure in the formula is mainly controlled by sodium phosphate dibasic, sodium phosphate monobasic and sodium chloride. The recombinant human lysozyme freeze-dried powder used in ZL 201810614434.9 patent adopts 5mM phosphate buffer + 0.25M sodium chloride for the last step of purification process, which introduces sodium phosphate monobasic, sodium phosphate dibasic and sodium chloride. When the recombinant human lysozyme freeze-dried powder is used to prepare artificial tears, the three of sodium phosphate monobasic, sodium phosphate dibasic and sodium chloride will interfere with the pH and osmotic pressure of the formula system at the same time. In the present application, the recombinant human lysozyme stock solution is eluted with 0.4% sodium chloride for the last step of purification process and subjected to sterilization filtration, only sodium chloride is introduced into the stock solution, avoiding the introduction of sodium phosphate monobasic and sodium phosphate dibasic, and when the stock solution is used to prepare artificial tears, hydrochloric acid can not be used for pH adjustment, and the pH system is more stable.

[0037] Osmotic pressure system stability: only sodium chloride is introduced into the recombinant human lysozyme stock solution and the content is fixed at 0.4%, when the artificial tears are prepared, the sodium chloride in the formula can be accurately calculated and the addition amount is fixed, which will not interfere with the osmotic pressure system of the artificial tears.

[0038] Suitable for large-scale production: it solves the uncertainty caused by the simultaneous change of pH and osmotic pressure in large-scale production, and provides support for stable large-scale production.

[0039] The novel artificial tears provided by the present application are prepared by the following steps:

[0040] (1) Weigh the required sodium hyaluronate and add it to the water for injection, stir to dissolve the sodium hyaluronate completely, and obtain a sodium hyaluronate solution.

[0041] (2) Add anhydrous disodium hydrogen phosphate, monobasic sodium phosphate monohydrate and sodium chloride to the sodium hyaluronate solution, stir to completely dissolve, and obtain a excipient solution.

[0042] (3) Add the recombinant human lysozyme stock solution to the excipient solution, stir to completely dissolve the recombinant human lysozyme stock solution, and obtain an un-determined volume new artificial tear solution.

[0043] (4) Add water for injection to the un-determined volume new artificial tear solution, and determine the volume to the theoretical weight, stir evenly, and obtain a new artificial tear solution.

[0044] (5) Filter the new artificial tear solution to remove bacteria.

[0045] The above preparation method can be carried out according to the following specific operation steps:

[0046] (1) Preparation of sodium hyaluronate solution

[0047] Weigh the required weight of sodium hyaluronate, slowly and uniformly add it to 60-80℃ water for injection, mechanically stir at 450-750 rpm for 2.5h or more, and naturally cool to room temperature to obtain a sodium hyaluronate solution.

[0048] (2) Usage calculation

[0049] Recombinant human lysozyme stock solution usage calculation: query the recombinant human lysozyme content in the COA report of the recombinant human lysozyme stock solution of the use batch, and calculate the usage of the recombinant human lysozyme stock solution according to the artificial tear preparation specification.

[0050] Sodium chloride usage calculation: multiply the new artificial tear preparation specification by the weight proportion of sodium chloride in the artificial tear, calculate the total amount of sodium chloride, and then calculate the mass of sodium chloride in the recombinant human lysozyme stock solution according to the usage of the recombinant human lysozyme stock solution in the previous step. Subtract the mass of sodium chloride in the recombinant human lysozyme stock solution from the total mass of sodium chloride to obtain the remaining sodium chloride usage.

[0051] (3) Preparation of excipient solution

[0052] Add anhydrous disodium hydrogen phosphate, monobasic sodium phosphate monohydrate and sodium chloride to the sodium hyaluronate solution, stir to completely dissolve, and obtain a excipient solution.

[0053] (4) Preparation of artificial tear

[0054] Add the calculated recombinant human lysozyme stock solution to the excipient solution, mechanically stir at 150-250 rpm for not less than 10 min, until completely dissolved, and obtain an un-determined volume artificial tear.

[0055] (5) Add water for injection to the new type of artificial tears, and then add water for injection to make up to the theoretical weight, and then mechanically stir at 150-250 rpm for not less than 10 min until completely dissolved to obtain the new type of artificial tears, and then transfer to the material storage tank.

[0056] (6) Filtration and sterilization

[0057] The filtration and sterilization method is used for sterilizing the artificial tears; preferably, the 0.22 μm sterilization filter is used for secondary filtration and sterilization; the filtration operation pressure used for filtration and sterilization can be controlled to be 0.35-0.40 MPa. After filtration and sterilization, the sterile artificial tears are transported to the filling equipment.

[0058] (7) Filling

[0059] The blow-filling-seal three-in-one sterile filling technology is used for filling. The prepared sterile medicinal liquid is filled into the container and sealed through the blow-filling-seal integrated machine under the protection of the A-level laminar flow, and the filling specification can be determined by the person skilled in the art according to the actual needs, for example, the filling specification is 0.4-0.8 mL / branch. The bottle blowing, medicinal liquid filling, and preparation sealing are completed online.

[0060] In the above steps, all the pipelines used for material transportation must be sterilized in advance.

[0061] The new type of artificial tears of the application is a topical sterile preparation, and the recombinant human lysozyme is an active protein. After the raw and auxiliary materials are prepared, the terminal filtration method is selected for sterilization, so that the activity of the recombinant human lysozyme can be guaranteed while ensuring the sterility of the artificial tears, and the inactivation of the lysozyme caused by high-temperature sterilization can be effectively avoided.

[0062] Compared with the prior art, the technical scheme of the application has the following beneficial effects:

[0063] The application unexpectedly finds that the new type of artificial tears containing a low concentration of recombinant human lysozyme has better repair and anti-inflammatory effects. Compared with the high-concentration group, the low-concentration group has a very significant effect of increasing tear secretion, promoting corneal repair, and resisting inflammation, can significantly promote the repair of human corneal epithelial cells, significantly reduce the content of inflammatory factors in the human corneal cell dry model, significantly reduce the content of inflammatory factors in the corneal epithelium of the rat corneal dryness model, and has significant repair and anti-inflammatory efficacy.

[0064] The novel artificial tear liquid containing recombinant human lysozyme of the application has a lower content of recombinant human lysozyme than the artificial tear liquid in ZL 201810614434.9, but has a more significant effect of increasing tear secretion and relieving inflammation, and can significantly reduce the content of IL-1beta, TNF-alpha and IL-6 inflammatory factors in the corneal epithelium of a rat atropine corneal xerosis model, and has better anti-inflammatory efficacy. The raw material of the recombinant human lysozyme in the application is derived from a recombinant human lysozyme stock solution, the purity of the recombinant human lysozyme is improved to 97.0%, the purity of the main component is improved, and the quality and safety are improved. Moreover, the artificial tear liquid preparation process of the application does not need to adjust the pH and the osmotic pressure molar concentration value, solves the uncertainty caused by the simultaneous change of the two variables of pH and osmotic pressure in large-scale production, greatly simplifies the large-scale (30 kg and above) production process, and improves the stability of large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 Results of cytotoxicity of recombinant human lysozyme on human corneal epithelial cells in Example 6.

[0066] Figure 2 Results of cytotoxicity of recombinant human lysozyme on human corneal epithelial cells in Example 6.

[0067] Figure 3 Results of ELISA detection of inflammatory factors of recombinant human lysozyme on human corneal epithelial cells in Example 6.

[0068] Figure 4 Results of fluorescence quantitative PCR detection of inflammatory factors of recombinant human lysozyme on human corneal epithelial cells in Example 6.

[0069] Figure 5 Results of cell immunoblotting detection of recombinant human lysozyme on human corneal epithelial cells in Example 6.

[0070] Figure 6 Results of the influence of artificial tear liquid on fluorescein sodium staining score of a rat corneal xerosis model in Example 7.

[0071] Figure 7 Results of the influence of artificial tear liquid on tear film break-up time of a rat corneal xerosis model in Example 7.

[0072] Figure 8 Results of the influence of artificial tear liquid on tear secretion of a rat corneal xerosis model in Example 7.

[0073] Figure 9 Results of the influence of artificial tear liquid on pathology of a rat corneal xerosis model in Example 7.

[0074] Figure 10The results of the effect of artificial tears on the inflammation factor score of the corneal xerosis of the rats in Example 7.

[0075] Figure 11 The results of the effect of artificial tears on the IL-1β immunohistochemistry of the corneal xerosis of the rats in Example 7.

[0076] Figure 12 The results of the effect of artificial tears on the TNF-α immunohistochemistry of the corneal xerosis of the rats in Example 7.

[0077] Figure 13 The results of the effect of artificial tears on the IL-6 immunohistochemistry of the corneal xerosis of the rats in Example 7.

[0078] Figure 14 The results of the effect of artificial tears on the IL-17 immunohistochemistry of the corneal xerosis of the rats in Example 7.

[0079] Figure 15 The results of the effect of artificial tears on the inflammation index of the corneal xerosis of the rats in Example 7. DETAILED DESCRIPTION

[0080] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it should not be understood as limiting the scope of the present application.

[0081] Preparation of the recombinant human lysozyme stock solution in Example 1

[0082] Reference is made to the preparation of the recombinant human lysozyme in Patent No. ZL201810614434.9.

[0083] The main component used in the present application, the recombinant human lysozyme stock solution, differs from that in ZL201810614434.9 in that, after the eluate is purified by chromatography with Sephadex G25 gel in 3.2.3 part 0099 of ZL201810614434.9, 0.4% sodium chloride with a mass concentration is added as a mobile phase to replace the buffer solution, a replacement solution is obtained, and the obtained replacement solution is then filtered with 0.22um to remove bacteria, thereby obtaining the recombinant human lysozyme stock solution. The recombinant human lysozyme stock solution meets the quality standards of the recombinant human lysozyme stock solution.

[0084] Quality standards and detection of the recombinant human lysozyme stock solution in Example 2

[0085] The quality standards of the recombinant human lysozyme stock solution are shown in Table 1.

[0086] Table 1 Quality standards of the recombinant human lysozyme stock solution

[0087]

[0088]

[0089] Type inspection item, detected once every half year, detected once every year.

[0090] Compared with ZL201810614434.9, the quality standard of the recombinant human lysozyme stock solution in the application does not include drying loss, ignition residue, total protein content, and endotoxin detection items. The same detection items as the recombinant human lysozyme freeze-dried powder are: identification-immunoblotting, pH, heavy metals, molecular weight, residual foreign DNA, residual yeast engineering bacteria protein, residual organic reagent, ultraviolet absorption, peptide map, isoelectric point, N-terminal amino acid sequencing, specific activity, and microbial limit.

[0091] The detection items different from the recombinant human lysozyme freeze-dried powder in ZL201810614434.9 are:

[0092] 1. Appearance

[0093] The recombinant human lysozyme stock solution in the application is a colorless and clear liquid.

[0094] 2. Recombinant human lysozyme content

[0095] According to the high-performance liquid chromatography method in the fourth part of Chinese Pharmacopoeia 2015, the recombinant human lysozyme content in the recombinant human lysozyme stock solution in the application is not less than 3 mg / mL by external standard method. The detection value of this item is related to the active ingredient content in the finished product of the new artificial tear.

[0096] 3. Purity

[0097] The purity of the recombinant human lysozyme stock solution in the application is detected by the fifth method in the fourth part of Chinese Pharmacopoeia 2020, and the purity of the recombinant human lysozyme is greater than or equal to 97.0%.

[0098] The recombinant human lysozyme stock solution in the application is determined by the area normalization method of the reverse-phase high-performance liquid chromatography method in the fourth part of Chinese Pharmacopoeia 2020. The loading amount is not less than 5 μg, detected at a wavelength of 280 nm, the theoretical plate number calculated by the recombinant human lysozyme chromatographic peak is not less than 16,000, and the recombinant human lysozyme main peak area calculated by the area normalization method is not less than 97% of the total area.

[0099] The purity of the recombinant human lysozyme stock solution is increased from 95.0% of the recombinant human lysozyme freeze-dried powder to 97.0%.

[0100] 4. Sodium chloride content

[0101] The sodium chloride content of the recombinant human lysozyme stock solution in the application is determined by the content method of physiological sodium chloride solution in the second part of Chinese Pharmacopoeia 2020, and the sodium chloride content should be 90.0%-110% of the labeled amount. The labeled amount is 0.4%, that is, the sodium chloride content should be 0.36%-0.44%.

[0102] The sodium chloride content of the recombinant human lysozyme stock solution directly affects the osmotic pressure molar concentration value of the new artificial tear, and the osmotic pressure molar concentration value directly affects the use experience of the new artificial tear. The control of this test item in the recombinant human lysozyme stock solution is conducive to the control of the osmotic pressure molar concentration value in the new artificial tear.

[0103] Comparative study of lyophilized powder and stock solution in artificial tears of Example 3

[0104] In the recombinant human lysozyme lyophilized powder process, sodium phosphate monobasic, sodium phosphate dibasic and sodium chloride are introduced in the purification process, while only sodium chloride is introduced in the recombinant human lysozyme stock solution purification process, and the sodium chloride content is controlled by sodium chloride content detection, which improves the stability of the stock solution system. Now the interference of lyophilized powder and stock solution on pH and osmotic pressure in artificial tears is compared. The results are shown in Table 2:

[0105] Table 2 Comparative study of lyophilized powder and stock solution in artificial tears system

[0106]

[0107]

[0108] Note: The stock solution is calculated based on recombinant human lysozyme, and the water in the stock solution is calculated as water for injection.

[0109] From the results in Table 2, the measured pH value of the recombinant human lysozyme lyophilized powder control group was 6.75, and the measured osmotic pressure was 273.5 mOsmol / kg; the measured pH value of the recombinant human lysozyme lyophilized powder group was 6.71, and the measured osmotic pressure was 298.4 mOsmol / kg, both the pH value and the osmotic pressure fluctuated, the pH fluctuation was 0.21, and the pH theoretical value 6.5 was adjusted by hydrochloric acid. The fluctuation value of osmotic pressure is 24.9 mOsmol / kg, which fluctuates greatly. The measured pH value of the recombinant human lysozyme stock solution control group was 6.51, and the measured osmotic pressure was 268.4 mOsmol / kg; the measured pH value of the recombinant human lysozyme stock solution group was 6.53, and the measured osmotic pressure was 276.2 mOsmol / kg, the pH value had no fluctuation, the fluctuation value of osmotic pressure was 7.8 mOsmol / kg, the fluctuation was small, and the osmotic pressure fluctuation value of 7.8 mOsmol / kg could be supplemented by 0.4% sodium chloride in the stock solution, which could be quantitatively calculated, stable and controllable.

[0110] Study on the content of sodium chloride in the prescription of Example 4

[0111] Process 1 to Process 5, wherein the anhydrous disodium hydrogen phosphate-sodium dihydrogen phosphate monohydrate buffer is 10 mM, and the sodium chloride content is set at 0.90%, 0.85%, 0.80%, 0.75%, and 0.70%, respectively, to prepare a new artificial tear, and detect the pH, osmotic pressure, activity, and content, and analyze the differences.

[0112] The preparation information is shown in Table 3 and Table 4:

[0113] Table 3: Preparation of new artificial tear with different concentrations of sodium chloride and detection results

[0114]

[0115] Table 4: Information table of each component of 300 mL new artificial tear containing recombinant human lysozyme

[0116]

[0117]

[0118] Process 1 to Process 5, i.e., the new artificial tears corresponding to the sodium chloride contents of 0.90%, 0.85%, 0.80%, 0.75%, and 0.70% are detected, and the results show that the pH value is 6.47 to 6.53, which is stable; the activity value of recombinant human lysozyme is 111% to 124%, which is stable; the content value of recombinant human lysozyme is 0.40, 0.37, 0.38, 0.37, and 0.38 mg / mL, respectively, which is stable; and the osmotic pressure molar concentration value is 305.4, 290.1, 278.8, 269.0, and 249.9 mOsmol / kg, respectively. The sodium chloride content is 0.70%-0.80%. The optimal one is 0.80% sodium chloride as the added amount of sodium chloride in the prescription.

[0119] Meanwhile, the contribution of sodium chloride, anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, sodium hyaluronate, and recombinant human lysozyme stock solution in the prescription to the osmotic pressure molar concentration value is studied, and the results are shown in Table 5.

[0120] Table 5: Research results of the contribution of different components in the prescription to the osmotic pressure

[0121]

[0122] As can be seen from the results, the osmotic pressure molar concentration value mainly depends on the sodium chloride and phosphate in the prescription, among which the sodium chloride is the main osmotic pressure regulator, and the phosphate adjusts the pH and contributes to part of the osmotic pressure.

[0123] Example 5: Preparation of artificial tear

[0124] The novel artificial tears provided by the application are prepared by the following steps:

[0125] (1) Sodium hyaluronate is added to water for injection to completely dissolve the sodium hyaluronate, and a sodium hyaluronate solution is obtained.

[0126] (2) Anhydrous disodium hydrogen phosphate, monohydrate sodium dihydrogen phosphate and sodium chloride are added to the sodium hyaluronate solution to completely dissolve them, and a excipient solution is obtained.

[0127] (3) Recombinant human lysozyme stock solution is added to the excipient solution to completely dissolve the recombinant human lysozyme stock solution, and an undetermined volume of novel artificial tears is obtained.

[0128] (4) Water for injection is added to the undetermined volume of novel artificial tears to determine the volume to the theoretical preparation amount, and each component is completely dissolved to obtain novel artificial tears.

[0129] (5) The novel artificial tears are filtered and sterilized.

[0130] Taking the preparation of 30 kg of novel artificial tears as an example, the above preparation method can be carried out according to the following specific steps:

[0131] (1) Preparation of sodium hyaluronate solution

[0132] 30.0 g of sodium hyaluronate is slowly and uniformly added to 15 kg of water for injection, mechanically stirred at 600 rpm for 2.5 h, and naturally cooled to room temperature to obtain a sodium hyaluronate solution.

[0133] (2) Calculation of the amount of raw materials

[0134] The content of recombinant human lysozyme in the COA report of the batch of recombinant human lysozyme stock solution is queried, and the specification of the novel artificial tears on the preparation instruction sheet is queried, and the amount of recombinant human lysozyme stock solution is calculated according to the preparation amount of artificial tears.

[0135] The amount of recombinant human lysozyme stock solution: taking LYZ-2402003 batch of recombinant human lysozyme stock solution (density 1.0 g / mL) as an example, 30 kg of novel artificial tears (density 1.0 g / mL) with a specification of 0.4 mg / mL (content of recombinant human lysozyme) are prepared. The content of recombinant human lysozyme in LYZ-2402003 batch of recombinant human lysozyme stock solution is obtained by querying the COA report, which is 3.20 mg / mL. According to the calculation, 30 kg of novel artificial tears with a specification of 0.4 mg / mL require:

[0136] The amount of recombinant human lysozyme stock solution: 30000 g ÷ 1.0 g / mL × 0.4 mg / mL = 12000 mg of recombinant human lysozyme; 12000 mg ÷ 3.20 mg / mL × 1.0 g / mL = 3750 g of recombinant human lysozyme stock solution is required.

[0137] Sodium chloride feeding amount: According to the content of sodium chloride in the stock solution, the content of sodium chloride in LYZ-2402003 batch stock solution is 4.0 mg / mL.

[0138] Sodium chloride feeding amount = 30000 g ÷ 1.0 g / mL × 8 mg / mL - 3750 g ÷ 1.0 g / mL × 4.0 mg / mL = 225.0 g.

[0139] (3) Preparation of excipient solution

[0140] Anhydrous disodium hydrogen phosphate 15.88 g, monosodium phosphate monohydrate 25.86 g, and sodium chloride 225.0 g were weighed. Anhydrous disodium hydrogen phosphate, monosodium phosphate monohydrate and sodium chloride were sequentially added to the sodium hyaluronate solution, and mechanical stirring was carried out at 600 rpm for 15 min until complete dissolution to obtain the excipient solution.

[0141] (4) Preparation of artificial tears

[0142] 3750 g of the calculated recombinant human lysozyme stock solution was added to the excipient solution, and mechanical stirring was carried out at 200 rpm for 10 min until complete dissolution to obtain the un-determined volume artificial tears.

[0143] (5) Water for injection was added to the un-determined volume artificial tears, and the volume was determined to 30 kg of the theoretical preparation amount, and mechanical stirring was carried out at 200 rpm for 10 min to completely dissolve the components to obtain the new artificial tears, and the new artificial tears were transferred to the material storage tank.

[0144] (6) Filtration and sterilization

[0145] The new artificial tears were sterilized by filtration: secondary filtration and sterilization were carried out using a 0.22 μm sterilization filter. After filtration and sterilization, the sterile artificial tears were transported to the filling equipment.

[0146] (7) Filling

[0147] Blow-filling-seal three-in-one sterile filling technology was used for filling. The prepared sterile liquid was filled into the container and sealed under A-level laminar flow protection by the blow-filling-seal machine, and the filling specification was 0.4-0.8 mL / branch. The bottle blowing, liquid filling and preparation sealing were completed online.

[0148] In the above steps, all the pipelines for material transportation must be sterilized in advance.

[0149] Example 6 Safety and effectiveness evaluation

[0150] In order to study the safety and effectiveness of the main component recombinant human lysozyme in the application, the Hong Kong Polytechnic University was commissioned to conduct the safety and effectiveness evaluation experiment of human lysozyme.

[0151] (1) Experimental materials

[0152] See Table 6 for details:

[0153] Table 6 Experimental materials and grouping information table

[0154] No. Sample Name Concentration (mg / mL) Sample Source 1 Recombinant human lysozyme (2.76 x 10 -9 M) 0.00004 mg / mL Huikang Biological 2 Recombinant human lysozyme (2.76 x 10 -8 M) 0.0004 mg / mL Huikang Biological 3 Recombinant human lysozyme (2.76 x 10 -7 M) 0.004 mg / mL Huikang Biological 4 Recombinant human lysozyme (2.76 x 10 -6 M) 0.04 mg / mL Huikang Biological 5 Recombinant human lysozyme (2.76 x 10 -5 M) 0.4 mg / mL Huikang Biological 6 Recombinant human lysozyme (2.76 x 10 -4 M) 4 mg / mL Huikang Biological 7 Human corneal epithelial cells / Beina Biological 8 Sodium hyaluronate (positive group) 0.2％ Huaxi Biological 9 Blank control Physiological saline /

[0155] (2) Test scheme

[0156] 2.1 Cell toxicity experiment

[0157] Corneal epithelial cells were planted in a 96-well plate at a density of 1.0 x 10 4 cells / well, cultured at 37°C, 5% CO2 for 16 hours to form a single cell layer. After removing the culture medium and washing once with PBS, different concentrations of recombinant human lysozyme (2.76 x 10 -9 , 2.76 x 10 -8 , 2.76 x 10 -7 , 2.76 x 10 -6 , 2.76 x 10 -5 , 2.76 x 10 -4 M) were added to each well, with negative and positive control groups set up. Cultured at 37°C, 5% CO2 for 48 hours.

[0158] After removing the culture medium and washing once with PBS, 50 μl of MTT working solution (5 mg / mL) was added to each well, and after 4 hours of reaction at 37°C, 100 μl of DMSO was added to each well to dissolve the precipitate. The absorbance of each well at 570 nm was measured using a microplate reader, and the cell viability was calculated.

[0159] 2.2 Cell anti-inflammatory experiment

[0160] Corneal epithelial cells were planted in a 12-well plate at a density of 3 x 10 5 cells / well, cultured at 37°C, 5% CO2 for 16 hours to form a single cell layer. After removing the culture medium and washing once with PBS, 450 mOsm (hypertonic culture medium) or 312 mOsm (normal culture medium) was added to the corresponding wells, and cultured at 37°C, 5% CO2 for 24 hours to induce dry eye model. After removing the culture medium and washing once with PBS, different concentrations of recombinant human lysozyme (2.76 x 10 -6 , 2.76 x 10 -5 , 2.76 x 10 -4 M) were added to each well, with negative and positive control groups set up. Cultured at 37°C, 5% CO2 for 24 hours.

[0161] After the incubation, the cell culture supernatant was collected for ELISA detection of inflammation-related content (inflammatory factors IL-1β, matrix metalloproteinase MMP-9); the cells were collected for fluorescence quantitative PCR detection of inflammation-related content (inflammatory factors IL-1β, IL-6); the cells were collected for Western blot detection of epithelial cell tight junction protein ZO-1.

[0162] (3) Data processing

[0163] N = 4, *** p < 0.001 vs. Blank control, ** p < 0.01 vs. Blank control.

[0164] (4) Test results and evaluation

[0165] 4.1 Cytotoxicity

[0166] The cytotoxicity results are shown in Figure 1 and Figure 2 . Low concentration of recombinant human lysozyme (2.76 × 10 -9 M) can significantly promote the proliferation of corneal epithelial cells; high concentration of recombinant human lysozyme (2.76 × 10 -4 M) shows significant cytotoxicity to the proliferation of corneal epithelial cells. The rest of the concentration groups have no cytotoxicity.

[0167] 4.2 Anti-inflammatory detection

[0168] The results of inflammation-related ELISA detection are shown in Figure 3 . Recombinant human lysozyme groups (2.76 × 10 -4 , 2.76 × 10 -5 M) can significantly reduce the content of IL-6 and MMP-9 in human corneal epithelial cells, and can reduce the content of inflammatory factors in cells.

[0169] The results of inflammation-related fluorescence PCR detection are shown in Figure 4 . Recombinant human lysozyme groups (2.76 × 10 -5 , 2.76 × 10 -6 M) can significantly reduce the gene level of IL-1β and IL-6 in human corneal epithelial cells, and can reduce the mRNA level of inflammatory factors in cells.

[0170] The results of inflammation-related Western blot detection are shown in Figure 5 . Recombinant human lysozyme groups (2.76 × 10 -4 M) have no repair effect on corneal cells. Recombinant human lysozyme groups (2.76 × 10 -5 , 2.76 × 10 -6M) can significantly increase the level of tight junction protein in human corneal epithelial cells, and can promote corneal cell repair.

[0171] According to the test results of 4.1 and 4.2, the high concentration recombinant human lysozyme 2.76×10 -4 M(4mg / mL) has certain cytotoxicity, does not have the effect of repairing corneal cells, and has significant anti-inflammatory effect; the medium concentration recombinant human lysozyme 2.76×10 -5 M(0.4mg / mL) has no cytotoxicity, has significant effect of promoting corneal cell repair, and has significant anti-inflammatory advantage; the low concentration recombinant human lysozyme 2.76×10 -6 M(0.04mg / mL) has no cytotoxicity, has significant effect of promoting corneal cell repair, and has significant anti-inflammatory effect, but the anti-inflammatory effect is not as good as that of the recombinant human lysozyme 2.76×10 -5 M(0.4mg / mL) group.

[0172] From the perspective of anti-inflammatory repair, the recombinant human lysozyme 2.76×10 -5 M(0.4mg / mL) group has the best advantage.

[0173] Pharmacodynamic evaluation of a new type of artificial tears containing recombinant human lysozyme

[0174] In order to prove the effectiveness of the new type of artificial tears containing recombinant human lysozyme, the pharmacodynamic experiment was entrusted to Shanghai Pharmaceutical Industry Research Institute Co., Ltd.

[0175] (1) Experimental materials

[0176] This embodiment verifies the curative effect and dose-effect relationship of the new type of artificial tears containing recombinant human lysozyme on keratosis sicca by using the keratosis sicca model of rats caused by subcutaneous injection of scopolamine for 10 days.

[0177] This experiment was completed in the pharmacology center of Shanghai Pharmaceutical Industry Research Institute Co., Ltd., and all animal experiments will comply with the experimental scheme and be approved by the animal ethics committee.

[0178] The information of the test sample and the control sample is shown in Table 7, and the preparation method of the artificial tears in Table 7 is the same as the preparation method of the artificial tears in Example 5, except for special instructions. The component content of each group is shown in Table 8.

[0179] Table 7 Information of test sample and control sample

[0180]

[0181] Table 8 Preparation component content table

[0182]

[0183] Note: " / " means not applicable.

[0184] [1] 0.1875 g is the requirement of human lysozyme in each kg of new artificial tears. The specific amount of human lysozyme stock solution used needs to be calculated according to the COA report. Similarly, the new artificial tears listed in other groups in the table are also the requirement of human lysozyme, and the specific amount of human lysozyme stock solution used needs to be calculated according to the COA report.

[0185] The main instruments and reagents are shown in Table 9.

[0186] Table 9 Main instrument and reagent information table

[0187] Instrument / reagent name Model / batch number Manufacturer Slit lamp microscope SLE-7 Chongqing Kanghuarui Ming Technology Hydrobromide scopolamine hydrochloride F2205450 Aladdin

[0188] The animals will be adapted to the environment before the experiment. The experimental animal information is shown in Table 10. The animals are raised in a barrier system, and the animal room environment conditions are controlled at room temperature 20-26℃, humidity 40-70%, and the feeding personnel record once in the morning and afternoon every day. The room is ventilated 10-20 times per hour. The lighting in the animal room is 12 / 12 hours light / dark cycle alternation; animals are raised in cages with 5-10 animals per cage. After entering the experiment, the animals are kept in group cages, and the animal cages are plastic cages made of polysulfone material with corn cob bedding. Each cage is identified with a cage card, and the animal body is marked as an internal identification number. The animals are free to eat, and the animals are free to drink from high-temperature and high-pressure sterilized tap water.

[0189] Table 10 Experimental animal information table

[0190] Species strain Wistar rats Grade SPF Number and gender 70 male Expected body weight at the beginning of administration 200g Certificate number 20220004049900 Purchased from Shanghai Sulei Experimental Animal Co., Ltd.

[0191] (2) Experimental scheme

[0192] 2.1 Experimental grouping

[0193] The animals were randomly divided into 7 groups according to their body weight one day before modeling, as shown in Table 11. They were blank control group, model group, Haier group, 1.5 mg / mL group, 0.75 mg / mL group, 0.375 mg / mL group, and 0.1875 mg / mL group.

[0194] Table 11 Animal grouping control information table

[0195]

[0196]

[0197] 2.2 Modeling

[0198] Except for the blank control group, the rats were subcutaneously injected with 0.6 mg / 0.1 mL hyoscine hydrobromide alternately in the hind limbs 4 times a day (8:00 and 11:00 in the morning, and 2:00 and 5:00 in the afternoon, respectively), and at the same time, the rats were placed in a ventilation device for continuous blowing for 12 hours a day for 10 days in succession, so as to prepare a corneal xerosis model.

[0199] 2.3 Grouping of rats before treatment

[0200] On the 3rd, 6th and 9th days of model induction, the degree of ocular lesions of the animals was evaluated by performing a sodium fluorescein score examination. According to the sodium fluorescein score, when the score of the animals in the model group reached 2 or above, the tear secretion examination (BUT score examination) and Schirmer I test were performed. According to the sodium fluorescein score as the main index, the tear secretion (BUT score examination) and Schirmer I test were considered, and the animals were evenly and randomly grouped, and the animals in each group were given drug intervention.

[0201] 2.4 Treatment and testing of rats

[0202] After the model rats were successfully prepared, the rats were grouped according to the model score value, and drug intervention was given, which was recorded as day 0. The following tests were performed on day 0, 3, 7, 10 and 14, respectively:

[0203] 1. BUT score examination; 2. Sodium fluorescein score examination; 3. Schirmer I test; 4. Corneal inflammation index score (pathology); 5. Corneal inflammation factor group staining.

[0204] (3) Data arrangement

[0205] The test results were compared with those of the model group and the blank control group, and the pharmacodynamic results of the artificial tears were analyzed. The data were arranged in Office Excel 2013, and the data were represented by One-way ANOVA was used for analysis; when two groups were compared, T-test was used for two-tailed test between the two groups, and when p<0.05, there was a significant difference between the two groups.

[0206] (4) Test results and evaluation

[0207] 4.1 Sodium fluorescein staining score examination

[0208] After the model rats were successfully prepared, the rats were grouped according to the model score value, and drug intervention was given, which was recorded as day 0, and the sodium fluorescein staining score examination was performed on day 0, 3, 7, 10 and 14, respectively.

[0209] Corneal fluorescein sodium staining score standard: after the drop of fluorescein sodium, 90 seconds later, the corneal epithelial fluorescein sodium staining was graded under the cobalt blue light of slit lamp microscope. The cornea was divided into 4 quadrants and scored respectively, and the final score was obtained by adding all the scores.

[0210] Score standard: no staining, 0 points; less than 30 point-like colorations, 1 point; more than 30 point-like colorations, but not diffuse, 2 points; severe diffuse staining, but no plaque-like staining, 3 points; plaque-like staining, 4 points.

[0211] The detection results are shown in Figure 6 , wherein, ##p<0.01 vs blank control group, **p<0.01 vs model group, *p<0.05 vs model group, aa p<0.01 vs haier group, a p<0.05 vs haier group, p<0.01 vs 1.5mg / mL group, p<0.05 vs 1.5mg / mL group. It can be seen from Figure 6 that the corneal fluorescein sodium staining scores of the artificial tears with a concentration of 0.375mg / mL were significantly different (p<0.01) from the model group, the haier group, the 0.75mg / mL group and the 1.5mg / mL group, and were significantly lower than those of the other groups.

[0212] 4.2 Breakup time of tear film examination

[0213] After the success of the rat model, the rats were grouped according to the model score value, and drug intervention was started, recorded as day 0, and the breakup time of tear film was examined on day 0, 3, 7, 10 and 14 respectively.

[0214] The results are shown in Figure 7 , wherein, ##p<0.01 vs blank control group. It can be seen from Figure 7 that the breakup time of tear film of the model group rats at different treatment times was reduced, and there was a significant difference (p<0.01) compared with the blank control group. Compared with the model group, there was no significant difference in the haier group, the 1.5mg / mL group, the 0.75mg / mL group, the 0.375mg / mL group and the 0.1875mg / mL group.

[0215] 4.3 Results of tear secretion examination

[0216] After the success of the rat model, the rats were grouped according to the model score value, and drug intervention was started, recorded as day 0, and the tear secretion examination was performed on day 0, 3, 7, 10 and 14 respectively.

[0217] Tear secretion detection: after the whole body anesthesia of the animal, the 1mm long end of the phenol red cotton line was placed on the lower eyelid near the outer canthus for 20 seconds, and the length of the red part of the cotton line was recorded.

[0218] Results are shown in Figure 8 Wherein, ##p<0.01 vs blank control group, **p<0.01 vs model group, *p<0.05 vs model group, aa p<0.01 vs Haier group, a p<0.05 vs Haier group, p<0.05 vs 1.5 mg / mL group. It can be seen from Figure 8 that the tear secretion of the model group rats was reduced, and compared with the blank control group, there was a significant difference (p<0.01). After 14 days of drug treatment in each group, the tear secretion of the model rats was lower than that of the blank control group, and there was a significant difference in statistics (p<0.01); after 10 days of drug treatment in each group, the tear secretion of the 0.375 and 0.1875 mg / mL groups was higher than that of the model group, and there was a difference in statistics (p<0.01 or p<0.05). The tear secretion of the 0.375 mg / mL new artificial tear group was higher than that of the model group, the Haier group and the 1.5 mg / mL group on the 14th day of treatment, and there was a significant difference in statistics (p<0.01 or p<0.05).

[0219] After 14 days of drug treatment in each group, the tear secretion of the 1.5, 0.75, 0.375 and 0.1875 mg / mL concentrations showed a dose-effect trend. With the decrease of concentration, the tear secretion increased, and reached the highest at 0.375 mg / mL. With the further decrease of concentration to 0.1875 mg / mL, the tear secretion decreased again. It showed that the effect of promoting tear secretion was best near 0.375 mg / mL concentration.

[0220] 4.4 Effect of artificial tears on the pathology of rat keratosis sicca model

[0221] After the rat model was successful, the rats were grouped according to the model score, and drug intervention was started, recorded as day 0, and H.E. staining was performed on day 0, 3, 7, 10 and 14.

[0222] The H.E. stained sections of rat corneas were graded under a light microscope, and the pathological grading standard was as follows:

[0223] Grade 0: The epithelial cell layer was complete, the flat cell layer was complete, and the stratification was clear. The basal cell layer was complete, arranged in order and closely attached to the BOWMAN membrane. The basal layer was regular and uniform.

[0224] Grade 1: The epithelial cell layer was complete, the flat cell layer was reduced, and the stratification was clear. The basal cell layer was complete, arranged in order and closely attached to the BOWMAN membrane. The basal layer was regular and uniform.

[0225] Grade 2: The epithelial cell layer was missing, the flat cell layer was reduced, and the stratification was chaotic. The basal cell layer was chaotic and incomplete, arranged irregularly. The basal layer had increased vacuoles.

[0226] Grade 3: epithelial cell layer has loss, flat cell layer loss, disorganized stratification. Basal cell layer loss, disorganized stratification incomplete, uneven arrangement. Basal layer vacuoles increased.

[0227] Compared with the blank control group, the pathological score of the model rats increased, and there was a significant difference (p<0.05). After 14 days of grouping and drug treatment, the pathological score of the model rats was higher than that of the blank control group, and there was a significant difference in statistics (p<0.05); the pathological score of the Hailu group, 1.5, 0.75, 0.375, 0.1875 mg / mL new artificial tear liquid group was lower than that of the model group, and there was a significant difference in statistics (p<0.05). The microscopic examination results show that compared with the model group, the Hailu group, the 1.5 mg / mL group and the 0.75 mg / mL group, the 0.375 mg / mL group shows the best treatment effect. The H.E. results are shown in Table 12.

[0228] Table 12 Effect of artificial tear on pathology of rat corneal xerosis model

[0229]

[0230]

[0231] 4.5 Artificial tear on rat corneal xerosis model immunohistochemical results

[0232] After the rat model was successful, the model was grouped according to the model score value, and drug intervention was started, recorded as day 0, paraffin embedding was performed at 14 days, sectioning, cytokine (IL-1β, IL-6, IL-17, TNF-α) H.E. immunohistochemical staining examination.

[0233] Light microscopic examination score: 0 grade: corneal epithelium without staining; 1 grade: only slight individual area staining of corneal epithelium; 2 grade: corneal epithelial basal cell layer staining, surface cells not stained; 3 grade: corneal epithelial cells are all stained.

[0234] Studies have shown that ocular surface epithelial cells express IL-1β, TNF-α, IL-6, IL-17 play a crucial role in the inflammatory response in dry eye. In dry eye animal model experiments, it was found that the new artificial tear of the application can significantly reduce the content of IL-1β, TNF-α, IL-6 inflammatory factors.

[0235] Results are shown in Figure 10 and Figures 11-14 . In Figure 10In the study, p<0.01 vs. blank control group, p<0.01 vs. model group, p<0.05 vs. model group, p<0.01 vs. Hylo group, p<0.05 vs. Hylo group, p<0.01 vs. 1.5 mg / mL group, and p<0.05 vs. 1.5 mg / mL group were observed. Figure 10 and Figures 11-14 In summary, compared with the control group, the model group showed significant increases in L-1β, TNF-α, and IL-6 (p<0.01). Compared with the model group, the Hylo group showed only a significant difference in IL-1β (p<0.05), while TNF-α, IL-6, and IL-17 showed no significant differences. Compared with the model group, the 0.375 mg / mL artificial tear group showed significant decreases in IL-1β, TNF-α, IL-6, and IL-17 (p<0.01). Compared with the Hylo group, the 0.375 mg / mL artificial tear group showed significant decreases in IL-1β, TNF-α, and IL-6 (p<0.01 or p<0.05). Compared with the 1.5 mg / mL group, the 0.375 mg / mL artificial tear group showed significant decreases in IL-1β, TNF-α, and IL-6 (p<0.01 or p<0.05). Figure 10 and Figures 11-14 In summary, at concentrations of 1.5, 0.75, 0.375, and 0.1875 mg / mL, IL-1β, TNF-α, and IL-6 exhibit a dose-response trend. As the concentration decreases, the levels of inflammatory factors IL-1β, TNF-α, and IL-6 decrease, reaching their lowest point at 0.375 mg / mL. However, as the concentration further decreases to 0.1875 mg / mL, the levels of inflammatory factors increase again. This indicates that the optimal anti-inflammatory concentration is around 0.375 mg / mL.

[0236] 4.6 Effects of artificial tears on inflammatory indices in a rat model of dry cornea

[0237] After successful rat modeling, rats were grouped according to their model scores and drug intervention was initiated, marked as day 0. Inflammatory factor indices were examined on days 0, 3, 7, 10, and 14.

[0238] Corneal inflammation index assessment was performed using a slit-light band, which consisted of three parts. Ciliary injection: No injection, 0 points; injection present, but less than 1 mm wide, 1 point; injection band wider than 2 mm, 3 points. Central corneal edema: No edema, 0 points; mild edema, iris texture still clear, 1 point; moderate edema, iris texture unclear, 2 points; iris and pupil not visible, 3 points. The scoring criteria for peripheral corneal edema were the same as for central corneal edema. The final corneal inflammation index score was the sum of the three scores divided by 9.

[0239] See results Figure 15Wherein, ##p<0.01 vs blank control group, **p<0.01 vs model group, *p<0.05 vs model group, aa p<0.01 vs Haier group, a p<0.05 vs Haier group, p<0.05 vs 1.5mg / mL group. Figure 15 It can be known that, compared with the blank control group, the inflammation indexes of the model group were significantly increased (p<0.01). Compared with the model group, the inflammation indexes of the Haier group at 3 and 10 days were significantly reduced (p<0.01 or p<0.05). Compared with the model group, the inflammation indexes of the 0.375mg / mL artificial tear group at 3, 7, 10 and 14 days were significantly reduced (p<0.01). Compared with the Haier group, the inflammation indexes of the 0.375mg / mL artificial tear group at 10 and 14 days were significantly reduced (p<0.01 or p<0.05). Compared with the 1.5mg / mL group, the inflammation indexes of the 0.375mg / mL artificial tear group at 10 and 14 days were significantly reduced (p<0.05).

[0240] It can be known from the result analysis that, after continuous treatment for 10 and 14 days, the inflammation indexes at the concentrations of 1.5, 0.75, 0.375 and 0.1875mg / mL showed a dose-effect trend, the inflammation indexes were reduced with the decrease of the concentration, and the inflammation indexes reached the lowest at 0.375mg / mL, and the inflammation indexes were increased again with the further decrease of the concentration to 0.1875mg / mL. It is indicated that the best anti-inflammatory concentration is near 0.375mg / mL.

[0241] The results show that the novel artificial tear of the application can reduce the ocular corneal injury of the scopolamine-induced corneal xerosis of a rat. After 14 days of eye drop treatment of 0.375mg / mL novel artificial tear, the tear secretion amount is higher than that of the model group, and compared with the Haier sodium hyaluronate eye drop group and other concentration groups, the effect is optimal. The novel artificial tear of the application can reduce the content of inflammatory factors in the corneal epithelium of the scopolamine-induced corneal xerosis model of a rat. The content of IL-1β, TNF-α and IL-6 inflammatory factors of the 0.375mg / mL novel artificial tear group is significantly reduced, and the effect is better than that of the Haier sodium hyaluronate eye drop group and other concentration groups.

[0242] The novel artificial tear of the application can reduce the inflammation index of the corneal epithelium of the scopolamine-induced corneal xerosis model of a rat. The effect of reducing the inflammation index of the corneal epithelium of the 0.375mg / mL novel artificial tear group is better than that of the Haier sodium hyaluronate eye drop group and other concentration groups.

[0243] Comprehensive evaluation of the difference between artificial tear groups, 0.375 mg / mL new artificial tears showed stable therapeutic effect at each observation time point and each index, and after 14 days of continuous treatment, its fluorescein sodium staining score, tear secretion, corneal epithelial IL1-β, TNF-α, IL-6, and inflammation index showed the best group, showing that it has the best effect in promoting corneal injury repair, increasing tear secretion, and limiting inflammation.

Claims

1. A novel artificial tear fluid containing recombinant human lysozyme, wherein, The new artificial tears include a main component and an auxiliary material, the main component is recombinant human lysozyme, and the content of the recombinant human lysozyme is 0.015% to less than 0.075% based on the total mass of the new artificial tears; The raw material of the recombinant human lysozyme is derived from a recombinant human lysozyme stock solution.

2. The novel artificial tears as claimed in claim 1 wherein, The content of the recombinant human lysozyme is 0.0375% to less than 0.075% based on the total mass of the new artificial tears.

3. The novel artificial tears as claimed in claim 1 wherein, The amino acid sequence of the recombinant human lysozyme is completely consistent with that of human natural lysozyme.

4. The novel artificial tears as claimed in claim 1 wherein, The recombinant human lysozyme stock solution is a colorless and clear liquid. Preferably, the content of sodium chloride in the recombinant human lysozyme stock solution is 0.36-0.44 w / v%. Preferably, the purity of the recombinant human lysozyme in the recombinant human lysozyme stock solution is greater than or equal to 97.0%. Preferably, the content of the recombinant human lysozyme in the recombinant human lysozyme stock solution is greater than or equal to 3 mg / mL. Preferably, the recombinant human lysozyme stock solution does not contain disodium hydrogen phosphate and sodium dihydrogen phosphate.

5. The novel artificial tear solution according to claim 1, wherein, The osmotic pressure molar concentration of the new artificial tears is 260 mOsmol / kg to less than 285 mOsmol / kg. Preferably, the osmotic pressure molar concentration of the new artificial tears is 270-280 mOsmol / kg.

6. The novel artificial tear fluid according to claim 1, wherein, The auxiliary material includes a pH stabilizer. Preferably, the pH stabilizer is a buffer solution composed of disodium hydrogen phosphate and sodium dihydrogen phosphate. More preferably, the disodium hydrogen phosphate is anhydrous disodium hydrogen phosphate, and the sodium dihydrogen phosphate is monohydrate sodium dihydrogen phosphate. Further preferably, the mass ratio of the anhydrous disodium hydrogen phosphate to the monohydrate sodium dihydrogen phosphate is (2.6-7.0):(7.0-11.2), and more preferably 5.3:8.

6.

7. The novel artificial tear fluid according to claim 1, wherein, The auxiliary material includes sodium hyaluronate. Preferably, the content of the sodium hyaluronate is 0.05-0.15% based on the total mass of the new artificial tears.

8. The novel artificial tears as claimed in claim 1 wherein, The auxiliary material includes sodium chloride. Preferably, the content of the sodium chloride is 0.70%-0.80% based on the total mass of the new artificial tears, and the content of the sodium chloride is calculated based on pure solid.

9. The novel artificial tear fluid according to claim 1, wherein, The pH value of the new artificial tears is 6.0-7.0, preferably 6.2-6.8, and more preferably 6.4-6.

6.

10. The novel artificial tear solution of claim 1, wherein, According to the total amount of the new artificial tears being 10000 parts by weight, the new artificial tears include: 1.5 parts to less than 7.5 parts of recombinant human lysozyme, 5.0-15.0 parts of sodium hyaluronate, 1.75-8.66 parts of anhydrous disodium hydrogen phosphate, 5.38-12.10 parts of monohydrate sodium dihydrogen phosphate, 70.0-80.0 parts of sodium chloride, and 9883.46-9909.65 parts of water.

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