A novel artificial tear containing recombinant human lysozyme

Abstract

The application provides a new artificial tear liquid containing recombinant human lysozyme, which comprises a main component and an excipient, wherein 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 tear liquid. It is found that the new artificial tear liquid containing a lower concentration of recombinant human lysozyme has better repair and anti-inflammatory effects, and the low concentration has a more significant effect of increasing tear secretion, promoting corneal repair and anti-inflammation compared with the high concentration. The raw material of the recombinant human lysozyme used in the application is derived from a recombinant human lysozyme stock solution, and the pH and the osmotic pressure molar concentration value do not need to be adjusted in the preparation process of the artificial tear liquid, so that the scale production process can be greatly simplified, and the stability of the scale production is improved.

Description

Technical Field

[0001] This invention relates to a novel artificial tear containing recombinant human lysozyme, belonging to the field of pharmaceutical technology. Background Technology

[0002] Dry eye syndrome is a general term for a variety of diseases caused by abnormal tear film quality or dynamics, leading to decreased tear film stability and accompanied by ocular discomfort and / or ocular surface lesions. In recent years, with the increasing popularity of electronic products and the high intensity of eye use, the prevalence of dry eye syndrome, including eye fatigue and eye inflammation, has been increasing. Statistics show that the number of dry eye patients in urban areas has been increasing rapidly at a rate of 10%-20% per year in recent years.

[0003] Human tears are a transparent liquid secreted by organs such as the lacrimal glands. They are composed of various substances such as inorganic salts, polysaccharides, proteins, and lipids, and have multiple functions such as barrier function, antibacterial, bactericidal, and immune regulation. They play an important role in protecting the eyeball, nourishing the ocular surface tissue, and improving visual function.

[0004] Currently, artificial tears on the market for dry eye caused by insufficient tear production due to various reasons mainly focus on basic fluid replenishment and moisturizing, and most contain chemical antibacterial agents. While these products can temporarily relieve dryness and discomfort, long-term exposure to antibacterial agents may cause allergies, inflammation, and other irritation, and may even lead to iatrogenic keratitis. Numerous studies have shown that antibacterial agents in eye drops have cytotoxic effects, affecting cell function and normal metabolism. Long-term use can damage the ocular surface tissues and reduce the eye's tolerance to eye drops. Some can even lead to persistent drug-induced conjunctivitis and drug-induced dry eye. Not only do they fail to solve the dry eye problem, they may actually worsen inflammation of the eye tissues.

[0005] The artificial tears described in ZL201810614434.9 require an additional step to adjust the pH in their preparation process. While this has little impact on small-scale production, it introduces significant interference during large-scale production (30 kg and above), lengthening the process cycle and increasing the complexity of the process. Summary of the Invention

[0006] The purpose of this invention is to provide a novel artificial tear containing recombinant human lysozyme and to optimize its large-scale production process. This novel artificial tear can effectively relieve and treat dry eye syndrome and has significant anti-inflammatory effects.

[0007] To achieve the above objectives, the present invention provides a novel artificial tear containing recombinant human lysozyme, comprising a main component and excipients, wherein the main component is recombinant human lysozyme, and the content of recombinant human lysozyme is 0.015% ≤ < 0.075% based on the total mass of the novel artificial tear.

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

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

[0010] According to a specific embodiment of the present invention, preferably, the amino acid sequence of the recombinant human lysozyme is completely identical to that of natural human lysozyme. The recombinant human lysozyme used in the present invention is obtained by microbial fermentation and has an amino acid sequence identical to that of natural human lysozyme, being 100% homologous.

[0011] The recombinant human lysozyme (rhLYZ, see patents ZL201810614434.9, ZL 201710736505.8, and ZL 201710233245.2) used in this invention can be obtained through microbial fermentation, preferably through fermentation and purification of engineered Pichia pastoris expressing human lysozyme. The molecular weight is 14700D. The amino acid sequence of the obtained recombinant human lysozyme is 100% identical to that of natural human lysozyme, indicating homology to humans. 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 invention, preferably, the raw material of the recombinant human lysozyme is derived from recombinant human lysozyme stock solution.

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

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

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

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

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

[0018] In this invention, the recombinant human lysozyme stock solution does not contain disodium hydrogen phosphate or sodium dihydrogen phosphate, that is, the content of disodium hydrogen phosphate and sodium dihydrogen phosphate is 0. If disodium hydrogen phosphate and sodium dihydrogen phosphate are present in the recombinant human lysozyme stock solution, it will cause pH fluctuations in the artificial tears when they are prepared.

[0019] According to a specific embodiment of the present invention, preferably, the excipients include a pH stabilizer.

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

[0021] In this invention, the mass ratio of anhydrous disodium hydrogen phosphate and sodium dihydrogen phosphate monohydrate can be within a certain range. Under this ratio, pH adjustment is not required during the large-scale preparation of the novel artificial tears. Other ratios will cause certain pH fluctuations during large-scale production, causing the pH to deviate from the set median value. In this case, a pH adjuster needs to be introduced for pH adjustment, which makes the large-scale preparation process of artificial tears more complicated.

[0022] According to a specific embodiment of the present invention, preferably, the osmolar concentration of the novel artificial tear is 260 mOsmol / kg ≤ 285 mOsmol / kg; more preferably, the osmolar concentration of the novel artificial tear is 270-280 mOsmol / kg.

[0023] The novel artificial tears described in this invention are slightly hypotonic. Patients with dry eye syndrome have a high ocular surface osmolality of tears. Supplementing with the novel slightly hypotonic artificial tears described in this invention can help restore the ocular surface osmolality to a normal isotonic state.

[0024] This invention proposes that ideal artificial tears should closely resemble natural tears in terms of composition, physicochemical properties, and biological functions. The ocular surface is directly exposed to the external environment, and healthy eyes continuously secrete tears, which, through blinking, spread to form a stable tear film. This tear film is the only barrier separating the ocular surface from the external environment. The biological function of natural tears is to protect the ocular surface; it is a comprehensive physiological function resulting from the interaction of its various components.

[0025] Patients with dry eye syndrome experience reduced tear production and increased evaporation, leading to the concentration of their natural tears, resulting in "hypertonic tears." From an osmotic perspective, artificial tears should be isotonic solutions. Therefore, the osmotic pressure of prescription artificial tears should be no higher than that of natural tears, or slightly lower, controlled to be mildly hypotonic.

[0026] According to a specific embodiment of the present invention, preferably, the excipients include sodium hyaluronate and / or sodium chloride.

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

[0028] According to a specific embodiment of the present invention, preferably, the sodium chloride content is 0.70%-0.80% based on the total mass of the novel artificial tears, and the sodium chloride content is based on pure solids; more preferably, the sodium chloride content in the novel artificial tears is 0.80% (including 0.4% sodium chloride contained in the recombinant human lysozyme stock solution).

[0029] According to a specific embodiment of the present invention, preferably, the pH value of the novel artificial tears is 6.0-7.0, more preferably 6.2-6.8, and even more preferably 6.4-6.6.

[0030] According to a specific embodiment of the present invention, preferably, based on a total amount of 10,000 parts by weight of the novel artificial tears, the novel artificial tears comprise: 1.5 parts ≤ recombinant human lysozyme addition < 7.5 parts, sodium hyaluronate 5.0-15.0 parts, anhydrous disodium hydrogen phosphate 1.75-8.66 parts, sodium dihydrogen phosphate monohydrate 5.38-12.10 parts, sodium chloride 70.0-80.0 parts, and water 9883.46-9909.65 parts.

[0031] In some specific embodiments of the present invention, based on a total amount of 10,000 parts by weight of the novel artificial tears, the novel artificial tears comprise: 4.0 parts of recombinant human lysozyme, 10.0 parts of sodium hyaluronate, 5.33 parts of anhydrous disodium hydrogen phosphate, 8.62 parts of sodium dihydrogen phosphate monohydrate, 80.0 parts of sodium chloride (including 0.4% sodium chloride from the recombinant human lysozyme stock solution), and 9,892.05 parts of water.

[0032] According to a specific embodiment of the present invention, preferably, based on a total amount of 10,000 parts by weight of the novel artificial tears, the novel artificial tears comprise: 3.75 parts of recombinant human lysozyme, 10.0 parts of sodium hyaluronate, 5.33 parts of anhydrous disodium hydrogen phosphate, 8.62 parts of sodium dihydrogen phosphate monohydrate, 80.0 parts of sodium chloride (including 0.4% sodium chloride from the recombinant human lysozyme stock solution), and 9,892.30 parts of water.

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

[0034] The preparation steps are the same as those in Example 1 of ZL 201810614434.9, which shows the preparation steps of recombinant human lysozyme. The difference is that in section 3.2.3, segment 0099 of Example 1 in ZL 201810614434.9, after the eluent is purified by chromatography using Sephadex G25 gel, 0.4% sodium chloride is added as the mobile phase for buffer replacement to obtain a replacement solution. The obtained replacement solution is then filtered through a 0.22 μm filter for sterilization to obtain the recombinant human lysozyme stock solution.

[0035] Compared with ZL 201810614434.9, the recombinant human lysozyme stock solution described in this invention has the following technical effects when applied to human lysozyme artificial tears:

[0036] The pH system is stable: Osmotic pressure changes in the formulation are mainly controlled by disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride. The recombinant human lysozyme lyophilized powder used in patent ZL 201810614434.9 uses a 5mM phosphate buffer + 0.25M sodium chloride solution in the final elution step of its purification process, introducing sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride. When using this recombinant human lysozyme lyophilized powder to prepare artificial tears, these three substances simultaneously interfere with the pH and osmotic pressure of the formulation system. In contrast, the recombinant human lysozyme stock solution used in this invention uses 0.4% sodium chloride for elution and sterilization filtration in the final step of its process. Only sodium chloride is introduced into the stock solution, avoiding the introduction of sodium dihydrogen phosphate and disodium hydrogen phosphate. When using this stock solution to prepare artificial tears, hydrochloric acid can be eliminated for pH adjustment, resulting in a more stable pH system.

[0037] Stable osmotic pressure system: The recombinant human lysozyme stock solution only introduces sodium chloride and the content is fixed at 0.4%. When preparing artificial tears, the sodium chloride in the formula can be accurately calculated and the amount added is fixed, which will not interfere with the osmotic pressure system of artificial tears.

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

[0039] The novel artificial tears provided by this invention are prepared using the following steps:

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

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

[0042] (3) Add recombinant human lysozyme stock solution to the excipient solution and stir to completely dissolve the recombinant human lysozyme stock solution to obtain a novel artificial tear solution with undiluted volume.

[0043] (4) Add water for injection to the undiluted new artificial tears, dilute to the theoretical weight, stir evenly, and obtain the new artificial tears.

[0044] (5) The new artificial tears are filtered and sterilized.

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

[0046] (1) Preparation of sodium hyaluronate solution

[0047] Weigh the required amount of sodium hyaluronate and slowly and evenly add it to water for injection at 60-80℃. Stir mechanically at 450-750rpm for more than 2.5 hours and allow it to cool naturally to room temperature to obtain a sodium hyaluronate solution.

[0048] (2) Usage Calculation

[0049] Calculation of Recombinant Human Lysozyme Stock Solution Usage: Check the recombinant human lysozyme content in the COA report of the batch of recombinant human lysozyme stock solution used. Based on the new artificial tear preparation specifications, calculate the usage of recombinant human lysozyme stock solution using the artificial tear preparation volume.

[0050] Sodium chloride usage calculation: Multiply the formulation specifications of the new artificial tears by the weight percentage of sodium chloride in the artificial tears to calculate the total amount of sodium chloride used. Then, calculate the mass of sodium chloride in the recombinant human lysozyme stock solution based on the amount used 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 amount of sodium chloride used.

[0051] (3) Preparation of excipient solution

[0052] Add anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, and sodium chloride to the sodium hyaluronate solution in sequence, and mechanically stir at 450-750 rpm for no less than 15 minutes until completely dissolved to obtain the excipient solution.

[0053] (4) Preparation of artificial tears

[0054] Add the calculated recombinant human lysozyme stock solution to the excipient solution, and mechanically stir at 150-250 rpm for no less than 10 minutes until completely dissolved to obtain undiluted artificial tears.

[0055] (5) Add water for injection to the undiluted new artificial tears, dilute to the theoretical weight, and mechanically stir at 150-250 rpm for no less than 10 minutes until completely dissolved to obtain the new artificial tears, and transfer to the material storage tank.

[0056] (6) Filtration and sterilization

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

[0058] (7) Filling

[0059] Aseptic filling technology combining blow-fill-seal is used for filling. The prepared aseptic solution is filled into containers and sealed under Class A laminar flow protection using a blow-fill-seal integrated machine. The filling specification can be determined by those skilled in the art based on actual needs; for example, a filling specification of 0.4-0.8 mL / vial. The production processes, including bottle blow molding, solution filling, and formulation sealing, are completed online.

[0060] In all the above steps, all pipes used for material transport must be sterilized beforehand.

[0061] The novel artificial tears of this invention are a sterile preparation for topical application. The recombinant human lysozyme is an active protein. After the raw materials and excipients are prepared, terminal filtration is used for sterilization. This ensures the sterility of the artificial tears while also maintaining the activity of the recombinant human lysozyme, effectively avoiding the inactivation of lysozyme that is easily caused by high-temperature sterilization.

[0062] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0063] This invention unexpectedly revealed that a novel artificial tear containing a lower concentration of recombinant human lysozyme exhibits better repair and anti-inflammatory effects. Compared to the high-concentration group, the low-concentration group significantly increases tear secretion, promotes corneal repair, and has anti-inflammatory effects. It can significantly promote the repair of human corneal epithelial cells; significantly reduce the content of inflammatory factors in a human corneal dryness model; and significantly reduce the content of inflammatory factors in the corneal epithelium of a rat model of corneal dryness, demonstrating significant repair and anti-inflammatory efficacy.

[0064] This invention discloses a novel artificial tear containing recombinant human lysozyme. Compared to the artificial tear in ZL 201810614434.9, it contains a lower amount of recombinant human lysozyme but exhibits a significant increase in tear secretion and a soothing, anti-inflammatory effect. It can significantly reduce the levels of IL-1β, TNF-α, and IL-6 inflammatory factors in the corneal epithelium of a rat model of scopolamine-induced keratoconjunctivitis, demonstrating good anti-inflammatory efficacy. The recombinant human lysozyme used in this invention is derived from recombinant human lysozyme stock solution, with a purity increased to 97.0%, improving the purity of the main component and enhancing quality and safety. Furthermore, the artificial tear preparation process of this invention eliminates the need for pH and osmotic pressure molar concentration adjustments, resolving the uncertainty caused by simultaneous changes in both pH and osmotic pressure during large-scale production. This greatly simplifies the large-scale (30 kg and above) production process and improves the stability of large-scale production. Attached Figure Description

[0065] Figure 1 The results of the cytotoxicity of recombinant human lysozyme on human corneal epithelial cells in Example 6 are shown.

[0066] Figure 2 The results of the cytotoxicity of recombinant human lysozyme on human corneal epithelial cells in Example 6 are shown.

[0067] Figure 3 The results of ELISA detection of inflammatory factors in human corneal epithelial cells by recombinant human lysozyme in Example 6 are shown.

[0068] Figure 4 The results of quantitative real-time PCR detection of inflammatory factors in human corneal epithelial cells by recombinant human lysozyme in Example 6 are shown.

[0069] Figure 5 The results of Western blot analysis of human corneal epithelial cells by recombinant human lysozyme in Example 6 are shown.

[0070] Figure 6 The results of the effect of artificial tears on the sodium fluorescein staining score of a rat model of dry cornea in Example 7 are shown.

[0071] Figure 7 The results show the effect of artificial tears on tear film breakup time in a rat model of dry cornea in Example 7.

[0072] Figure 8 The results of the effect of artificial tears on tear secretion in rats with dry cornea in Example 7 are shown.

[0073] Figure 9 The results show the effects of artificial tears on the pathology of a rat model of dry cornea in Example 7.

[0074] Figure 10The results of the effect of artificial tears on the inflammatory factor scores of rats with dry cornea in Example 7.

[0075] Figure 11 The immunohistochemical results of artificial tears in rats with dry cornea and IL-1β in Example 7 are shown.

[0076] Figure 12 The results of immunohistochemical examination of artificial tears in rats with xerokeratosis TNF-α in Example 7 are shown.

[0077] Figure 13 The immunohistochemical results of artificial tears on IL-6 in rats with dry cornea in Example 7.

[0078] Figure 14 The immunohistochemical results of artificial tears on IL-17 in rats with dry cornea in Example 7 are shown.

[0079] Figure 15 The results of the effect of artificial tears on the inflammation index of dry cornea in rats in Example 7. Detailed Implementation

[0080] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0081] Example 1: Preparation of Recombinant Human Lysozyme Stock Solution

[0082] The preparation of recombinant human lysozyme is cited in patent number ZL201810614434.9.

[0083] The main component used in this invention, recombinant human lysozyme, is recombinant human lysozyme stock solution. The difference lies in the following: In section 3.2.3, segment 0099 of ZL201810614434.9, after purifying the eluent using Sephadex G25 gel chromatography, 0.4% sodium chloride is added as the mobile phase for buffer replacement, yielding a replacement solution. This replacement solution is then sterilized by 0.22µm filtration, thus obtaining the recombinant human lysozyme stock solution. This meets the quality standards for recombinant human lysozyme stock solution.

[0084] Example 2: Quality Standards and Testing of Recombinant Human Lysozyme Stock Solution

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

[0086] Table 1 Quality Standards for Recombinant Human Lysozyme Stock Solution

[0087]

[0088]

[0089] * indicates type testing items, * to be tested every six months, ** to be tested annually.

[0090] Compared to ZL201810614434.9, the quality standard for recombinant human lysozyme stock solution in this invention does not include tests for loss on drying, residue on ignition, total protein content, or endotoxins. The same test items as for recombinant human lysozyme lyophilized powder include: identification-immunoblotting, pH, heavy metals, molecular weight, exogenous DNA residue, residual yeast cell protein, residual organic reagents, UV absorption, peptide mapping, isoelectric point, N-terminal amino acid sequencing, specific activity, and microbial limits.

[0091] The detection items that differ from those in recombinant human lysozyme lyophilized powder in ZL201810614434.9 are:

[0092] 1. Characteristics

[0093] In this invention, the recombinant human lysozyme stock solution is a colorless and clear liquid.

[0094] 2. Recombinant human lysozyme content

[0095] In this invention, the recombinant human lysozyme stock solution, calculated using the external standard method according to General Chapter 0512 of the Chinese Pharmacopoeia, has a recombinant human lysozyme content of not less than 3 mg / mL. This detection value is related to the content of the active ingredient in the finished novel artificial tears.

[0096] 3. Purity

[0097] The recombinant human lysozyme stock solution of this invention, when tested according to Method 5 of General Chapter 0541 in Part IV of the 2020 edition of the Chinese Pharmacopoeia, has a purity of ≥97.0%.

[0098] In this invention, the recombinant human lysozyme stock solution was determined according to the area normalization method of reversed-phase high-performance liquid chromatography (RP-HPLC) in General Chapter 0512 of the 2020 edition of the Chinese Pharmacopoeia, Part IV. The sample loading amount was not less than 5 μg, and the detection was performed at a wavelength of 280 nm. The theoretical step number calculated based on the recombinant human lysozyme chromatographic peak was not less than 16,000. Calculated using the area normalization method, the area of ​​the main peak of the recombinant human lysozyme was not less than 97% of the total area.

[0099] The purity of the recombinant human lysozyme stock solution was increased from 95.0% to 97.0% of the recombinant human lysozyme lyophilized powder.

[0100] 4. Sodium chloride content

[0101] In this invention, the recombinant human lysozyme stock solution, according to the physiological sodium chloride solution content method in the 2020 edition of the Chinese Pharmacopoeia, Part II, should have a sodium chloride content of 90.0%-110% of the labeled amount. The labeled amount is 0.4%, which means the sodium chloride content should be 0.36%-0.44%.

[0102] The sodium chloride content of recombinant human lysozyme stock solution directly affects the osmolality of novel artificial tears, which in turn directly impacts the user experience. Controlling this component in the recombinant human lysozyme stock solution is beneficial for controlling the osmolality of novel artificial tears.

[0103] Example 3: Comparative Study of Lyophilized Powder and Stock Solution in Artificial Tears

[0104] In the purification process of recombinant human lysozyme lyophilized powder, sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride are introduced, while in the purification process of recombinant human lysozyme stock solution, only sodium chloride is introduced. Furthermore, the sodium chloride content in the recombinant human lysozyme stock solution is controlled by sodium chloride content detection, thus improving the stability of the stock solution system. A comparative study is conducted on the interference of lyophilized powder and stock solution on pH and osmotic pressure in artificial tears. The results are shown in Table 2.

[0105] Table 2 Comparative Study of Lyophilized Powder and Stock Solution in Artificial Tear System

[0106]

[0107]

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

[0109] As shown 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 pH value and osmotic pressure fluctuated. The pH fluctuation was 0.21, requiring adjustment to the theoretical pH value of 6.5 using hydrochloric acid. The osmotic pressure fluctuation was 24.9 mOsmol / kg, indicating significant fluctuation. The recombinant human lysozyme stock solution control group had a measured pH of 6.51 and a measured osmotic pressure of 268.4 mOsmol / kg; the recombinant human lysozyme stock solution group had a measured pH of 6.53 and a measured osmotic pressure of 276.2 mOsmol / kg. The pH value remained stable, while the osmotic pressure fluctuation was 7.8 mOsmol / kg. The fluctuation was small, and the 7.8 mOsmol / kg osmotic pressure fluctuation could be compensated by the 0.4% sodium chloride in the stock solution. This fluctuation was quantifiable, stable, and controllable.

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

[0111] Processes ① through ⑤ were set up, in which the anhydrous disodium hydrogen phosphate-sodium dihydrogen phosphate monohydrate buffer solution was 10 mM, and the sodium chloride content was set to 0.90%, 0.85%, 0.80%, 0.75%, and 0.70%, respectively, to prepare a new type of artificial tear solution. The pH, osmotic pressure, activity, and content were tested, and the differences were analyzed.

[0112] For detailed preparation information, please refer to Tables 3 and 4:

[0113] Table 3. Preparation of novel artificial tears with different concentrations of sodium chloride and test results.

[0114]

[0115] Table 4. Information on the components of a novel artificial tear solution containing recombinant human lysozyme, prepared in 300 mL.

[0116]

[0117]

[0118] Processes ① to ⑤, corresponding to sodium chloride contents of 0.90%, 0.85%, 0.80%, 0.75%, and 0.70%, were tested. Results showed: pH values ​​ranged from 6.47 to 6.53, indicating stable pH; recombinant human lysozyme activity ranged from 111% to 124%, indicating stable activity; recombinant human lysozyme content was 0.40, 0.37, 0.38, 0.37, and 0.38 mg / mL, indicating stable content; and osmolality was 305.4, 290.1, 278.8, 269.0, and 249.9 mOsmol / kg, respectively. Sodium chloride content of 0.70%-0.80% is acceptable. Optimal, 0.80% sodium chloride was selected as the amount of sodium chloride added in the formulation.

[0119] The contributions of sodium chloride, anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, sodium hyaluronate, and recombinant human lysozyme stock solution to the osmolar concentration of the formulation were also studied, and the results are shown in Table 5.

[0120] Table 5. Results of the study on the contribution of different components in the formulation to osmotic pressure.

[0121]

[0122] The results show that the osmolar concentration value is mainly adjusted by sodium chloride and phosphate in the formulation. Sodium chloride is the main osmolar regulator, while phosphate not only adjusts the pH but also contributes to the osmolarity.

[0123] Example 5: Preparation of Artificial Tears

[0124] The novel artificial tears provided by this invention are prepared using the following steps:

[0125] (1) Add sodium hyaluronate to water for injection to completely dissolve the sodium hyaluronate and obtain a sodium hyaluronate solution.

[0126] (2) Add anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate and sodium chloride to the sodium hyaluronate solution and dissolve them completely to obtain the excipient solution.

[0127] (3) Add recombinant human lysozyme stock solution to the excipient solution to completely dissolve the recombinant human lysozyme stock solution and obtain an undiluted novel artificial tear solution.

[0128] (4) Add water for injection to the unconcentrated new artificial tears and bring the volume to the theoretical preparation volume to completely dissolve all components and obtain the new artificial tears.

[0129] (5) The new 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] Weigh 30.0g of sodium hyaluronate and slowly and evenly add it to 15kg of 70% water for injection. Stir mechanically at 600rpm for 2.5h and allow to cool naturally to room temperature to obtain a sodium hyaluronate solution.

[0133] (2) Calculation of material input

[0134] Check the recombinant human lysozyme content in the COA report of the batch of recombinant human lysozyme stock solution used, then check the specifications of the new artificial tears on the new artificial tear preparation instruction sheet, and finally calculate the amount of recombinant human lysozyme stock solution to be used based on the amount of artificial tears prepared.

[0135] Recombinant human lysozyme stock solution dosage: Taking batch LYZ-2402003 recombinant human lysozyme stock solution (density 1.0 g / mL) as an example, to prepare 30 kg of novel artificial tears with a specification of 0.4 mg / mL (content of recombinant human lysozyme) (density 1.0 g / mL), the COA report shows that the recombinant human lysozyme content in batch LYZ-2402003 is 3.20 mg / mL. Therefore, the following dosage is required to prepare 30 kg of novel artificial tears with a specification of 0.4 mg / mL:

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

[0137] Sodium chloride dosage: According to the raw material quality inspection report, the sodium chloride content of the raw material is 4.0 mg / mL in batch LYZ-2402003.

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

[0139] (3) Preparation of excipient solution

[0140] Weigh out 15.88 g of anhydrous disodium hydrogen phosphate, 25.86 g of sodium dihydrogen phosphate monohydrate, and 225.0 g of sodium chloride. Add the anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, and sodium chloride sequentially to the sodium hyaluronate solution, and mechanically stir at 600 rpm for 15 minutes until completely dissolved to obtain the excipient solution.

[0141] (4) Preparation of artificial tears

[0142] Add 3750g of the calculated recombinant human lysozyme stock solution to the excipient solution, and mechanically stir at 200rpm for 10min until completely dissolved to obtain undiluted artificial tears.

[0143] (5) Add water for injection to the undiluted artificial tears, dilute to the theoretical preparation volume of 30 kg, and mechanically stir at 200 rpm for 10 min to completely dissolve all components, thereby obtaining a new type of artificial tears, and transfer it to the material storage tank.

[0144] (6) Filtration and sterilization

[0145] The novel artificial tears were sterilized using a filtration sterilization method: a 0.22μm sterilization filter was used for two-stage filtration sterilization. After filtration sterilization, the sterile artificial tears were delivered to the filling equipment.

[0146] (7) Filling

[0147] The filling process utilizes a three-in-one aseptic filling technology combining blow-fill-seal. The prepared aseptic solution is filled into containers and sealed using a blow-fill-seal integrated machine under Class A laminar flow protection. The filling specification is 0.4-0.8 mL / vial. The entire production process, including bottle blow molding, solution filling, and formulation sealing, is completed online.

[0148] In all the above steps, all pipes used for material transport must be sterilized beforehand.

[0149] Example 6 Safety and effectiveness evaluation

[0150] To investigate the safety and efficacy of the main component, recombinant human lysozyme, in this invention, the Hong Kong Polytechnic University was commissioned to conduct safety and efficacy evaluation experiments on human lysozyme.

[0151] (1) Experimental materials

[0152] See Table 6 for details:

[0153] Table 6 Experimental Materials and Grouping Information

[0154] 1 <![CDATA[Recombinant human lysozyme (2.76×10 -9 M)]]> 0.00004 mg / mL Huikang Biotechnology 2 <![CDATA[Recombinant human lysozyme (2.76×10 -8 M)]]> 0.0004 mg / mL Huikang Biotechnology 3 <![CDATA[Recombinant human lysozyme (2.76×10 -7 M)]]> 0.004 mg / mL Huikang Biotechnology 4 <![CDATA[Recombinant human lysozyme (2.76×10 -6 M)]]> 0.04 mg / mL Huikang Biotechnology 5 <![CDATA[Recombinant human lysozyme (2.76×10 -5 M)]]> 0.4 mg / mL Huikang Biotechnology 6 <![CDATA[Recombinant human lysozyme (2.76×10 -4 M)]]> 4mg / mL Huikang Biotechnology 7 Human corneal epithelial cells / Beina Bio 8 Sodium hyaluronate (positive group) 0.2％ Bloomage Biotechnology 9 Blank control physiological saline /

[0155] (2) Test scheme

[0156] 2.1 Cytotoxicity assay

[0157] Corneal epithelial cells were 1.0 × 10⁻⁶ 4 Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured at 37°C and 5% CO2 for 16 hours to form a monolayer. After removing the culture medium and washing once with PBS, each well was then filled with a solution containing different concentrations of recombinant human lysozyme (2.76 × 10⁻⁶). -9 2.76×10 -8 2.76×10 -7 2.76×10 -6 2.76×10 -5 2.76×10 -4 The culture medium (M) was used, with both a negative control and a positive control group. The medium was incubated at 37°C and 5% CO2 for 48 hours.

[0158] After removing the culture medium and washing once with PBS, add 50 μl of MTT working solution (5 mg / mL) to each well and react at 37°C for 4 hours. Then, add 100 μl of DMSO to each well to dissolve the precipitate. Measure the absorbance of each well at 570 nm using a microplate reader and calculate cell viability.

[0159] 2.2 Cellular Anti-inflammatory Experiment

[0160] Corneal epithelial cells were 3 × 10 5 Cells were seeded at a density in 12-well plates and cultured at 37°C and 5% CO2 for 16 hours to form a monolayer. After removing the culture medium and washing once with PBS, 450 mOsm (hypertonic medium) or 312 mOsm (normal medium) was added to the corresponding wells, and the plates were incubated at 37°C and 5% CO2 for 24 hours to induce a dry eye model. After removing the culture medium and washing once with PBS, each well was then filled with a solution containing different concentrations of recombinant human lysozyme (2.76 × 10⁻⁶). -6 2.76×10 -5 2.76×10 -4 The culture medium (M) was used, with both a negative control and a positive control group. The medium was incubated at 37°C and 5% CO2 for 24 hours.

[0161] After incubation, the cell culture supernatant was collected for ELISA detection of inflammation-related contents (inflammatory cytokine IL-1β, matrix metalloproteinase MMP-9); cells were collected for quantitative real-time PCR detection of inflammation-related contents (inflammatory cytokines IL-1β, IL-6); and cells were collected for Western blotting detection (epithelial cell tight junction protein ZO-1).

[0162] (3) Data processing

[0163] The analysis was performed using a T-test, with N=4. *** indicates p<0.001 vs. Blank control, and ** indicates p<0.01 vs. Blank control.

[0164] (4) Test results and evaluation

[0165] 4.1 Cytotoxicity

[0166] Cytotoxicity results are shown in Figure 1 and Figure 2 Low concentration of recombinant human lysozyme (2.76 × 10⁻⁶) -9 M) can significantly promote corneal epithelial cell proliferation; high concentration of recombinant human lysozyme (2.76×10) -4 M) showed significant cytotoxicity against corneal epithelial cell proliferation. No cytotoxicity was observed in the other concentration groups.

[0167] 4.2 Anti-inflammatory test

[0168] Inflammation-related ELISA test results are shown below Figure 3 Recombinant human lysozyme group (2.76×10⁻⁶) -4 2.76×10 -5 M) can significantly reduce the levels of IL-6 and MMP-9 in human corneal epithelial cells, and can alleviate the levels of inflammatory factors in cells.

[0169] Results of inflammation-related fluorescent PCR detection are shown in Figure 4 Recombinant human lysozyme group (2.76×10⁻⁶) -5 2.76×10 -6 M) can significantly reduce the gene levels of IL-1β and IL-6 in human corneal epithelial cells and alleviate the mRNA levels of inflammatory factors in cells.

[0170] Results of Western blot analysis of inflammation-related cells are shown below. Figure 5 Recombinant human lysozyme group (2.76×10⁻⁶) -4 M) No corneal cell repair activity. Recombinant human lysozyme group (2.76 × 10⁻⁶) -5 2.76×10 -6M) can significantly increase the level of tight junction protein in human corneal epithelial cells, which can promote corneal cell repair.

[0171] Based on the combined results of experiments 4.1 and 4.2, the high concentration of recombinant human lysozyme is 2.76 × 10⁻⁶. -4 M (4 mg / mL) exhibits some cytotoxicity, lacks corneal cell repair activity, but possesses significant anti-inflammatory effects; a medium concentration of recombinant human lysozyme 2.76 × 10⁻⁶ -5 M (0.4 mg / mL) was non-cytotoxic, exhibited significant corneal cell repair-promoting effects, and also possessed significant anti-inflammatory advantages; low-concentration recombinant human lysozyme 2.76 × 10⁻⁶ -6 M (0.04 mg / mL) showed no cytotoxicity, significant corneal cell repair activity, and significant anti-inflammatory effects, but its anti-inflammatory efficacy was less than that of recombinant human lysozyme 2.76 × 10⁻⁶. -5 Group M (0.4 mg / mL).

[0172] Considering all factors from an anti-inflammatory and repair perspective, recombinant human lysozyme 2.76×10 -5 The M (0.4 mg / mL) group showed the best advantage.

[0173] Example 7: Pharmacodynamic evaluation of a novel artificial tear containing recombinant human lysozyme

[0174] To demonstrate the effectiveness of the novel artificial tears containing recombinant human lysozyme described in this invention, pharmacodynamic experiments were commissioned to Shanghai Institute of Pharmaceutical Industry Co., Ltd.

[0175] (1) Experimental materials

[0176] This embodiment uses a rat model of xerokeratosis induced by subcutaneous injection of scopolamine for 10 days to verify the efficacy and dose-response relationship of a novel artificial tear containing recombinant human lysozyme for xerokeratosis induced by ...

[0177] This experiment was conducted at the Pharmacology Center of Shanghai Pharmaceutical Industry Research Institute Co., Ltd. All animal experiments will adhere to the experimental protocol and be approved by the animal ethics committee.

[0178] Information on the test samples and reference samples is detailed in Table 7. Unless otherwise specified, the preparation method of the artificial tears in Table 7 is the same as that of the artificial tears in Example 5. The component contents of each group are shown in Table 8.

[0179] Table 7 Information on Test Samples and Reference Samples

[0180]

[0181] Table 8. Content of Components in the Formulation

[0182]

[0183] Note: " / " indicates that it is not applicable.

[0184] [1]: 0.1875g is the required amount of human lysozyme per kg of the new artificial tears. The specific amount of human lysozyme stock solution used needs to be calculated based on the COA report. Similarly, the required amount of human lysozyme in the other groups of new artificial tears listed in the table is also the required amount of human lysozyme. The specific amount of human lysozyme stock solution used needs to be calculated based on the COA report.

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

[0186] Table 9. Information on Major Instruments and Reagents

[0187] Slit lamp microscope SLE-7 Chongqing Kanghua Ruiming Technology Scopolamine hydrobromide F2205450 Aladdin

[0188] Animals will undergo environmental acclimatization before the experiment. Animal information is shown in Table 10. Animals will be housed in a barrier system, with room temperature controlled at 20℃-26℃ and humidity at 40%-70%. Records will be kept twice daily, once in the morning and once in the afternoon. Air exchange rate will be 10-20 times per hour. Lighting in the animal room will alternate between light and dark 12 / 12 hours. Animals will be housed in groups of 5-10 per cage. After enrollment, animals will remain in group cages made of polysulfone plastic with corncob bedding. Each cage will be identified by a cage tag, and animal body markings will serve as group identification. Animals will have free access to food and drinking water sterilized by high temperature and pressure.

[0189] Table 10 Information on Laboratory Animals

[0190] grade SPF Quantity and gender 70 males Expected body weight at the start of administration 200g Certificate Number 20220004049900 Purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0191] (2) Experimental scheme

[0192] 2.1 Experimental Grouping

[0193] The day before the modeling experiment, the animals were randomly divided into 7 groups based on their body weight, as detailed in Table 11. These groups were: blank control group, model group, Hydral 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 and Control Information

[0195]

[0196]

[0197] 2.2 Modeling

[0198] Except for the blank control group, rats were alternately injected subcutaneously with 0.6 mg / 0.1 mL scopolamine hydrobromide in their hind limbs four times a day (at 8:00 and 11:00 am, and at 2:00 and 5:00 pm, respectively). At the same time, the rats were placed in a ventilation device and exposed to air for 12 hours a day for 10 consecutive days to prepare a model of dry cornea.

[0199] 2.3 Grouping of rats before treatment

[0200] On days 3, 6, and 9 of model induction, fluorescein sodium scoring was performed to evaluate the degree of ocular lesions in the animals. Using the fluorescein sodium score as the standard, animals in the model group with a score of 2 or above underwent tear secretion testing (BUT scoring) and Schirmer I test. Based on the fluorescein sodium score as the primary indicator, and taking into account tear secretion (BUT scoring) and Schirmer I test, animals were randomly and uniformly grouped and given drug intervention according to their group.

[0201] 2.4 Rat Treatment and Testing

[0202] After successful rat model establishment, rats were stratified and grouped according to their model scores and given drug intervention, recorded as day 0. The following tests were performed on days 0, 3, 7, 10, and 14:

[0203] 1. BUT score test; 2. Fluorescein sodium score test; 3. Schirmer I test; 4. Corneal inflammation index score (pathology); 5. Histochemical staining of corneal inflammatory factors.

[0204] (3) Data processing

[0205] The test results were compared with those of the model group and the blank control group to analyze the pharmacodynamic results of artificial tears. Data are in Office Excel 2013. The results were presented in the table. One-way ANOVA was used for analysis; when comparing two groups, a two-tailed T-test was used to detect the difference between the two groups. A significant difference was considered to be between the two groups when p < 0.05.

[0206] (4) Test results and evaluation

[0207] 4.1 Fluorescein sodium staining scoring examination

[0208] After successful rat modeling, rats were grouped according to their model scores and drug intervention was initiated, marked as day 0. Fluorescein sodium staining scores were measured on days 0, 3, 7, 10, and 14.

[0209] Corneal fluorescein staining scoring criteria: After instilling fluorescein, corneal epithelial fluorescein staining and grading are performed under cobalt blue light of a slit-lamp microscope 90 seconds later. The cornea is divided into 4 quadrants and scored separately. All scores are added together to obtain the final score.

[0210] Scoring criteria: No staining, 0 points; fewer than 30 punctate staining spots, 1 point; more than 30 punctate staining spots, but not diffuse, 2 points; severe diffuse staining, but no patchy staining, 3 points; patchy staining, 4 points.

[0211] Test results are shown Figure 6 Among them, ##p<0.01 vs blank control group, **p<0.01 vs model group, *p<0.05 vs model group, aa p<0.01 vs Hylo group, ap<0.05 vs Hylo group, p<0.01 vs 1.5mg / mL group, and p<0.05 vs 1.5mg / mL group. Figure 6 It can be seen that, after 10 and 14 days of artificial tear administration, compared with the model group, Hylo group, and 0.75 mg / mL and 1.5 mg / mL groups, the corneal fluorescein staining score of artificial tear at a concentration of 0.375 mg / mL was significantly different (p<0.01), and was significantly lower than other groups.

[0212] 4.2 Tear film breakup time examination

[0213] After successful rat modeling, rats were grouped according to their model scores and drug intervention was initiated, marked as day 0. Tear film breakup time was examined on days 0, 3, 7, 10, and 14.

[0214] See results Figure 7 Among them, ##p<0.01 vs blank control group. (By...) Figure 7 It was found that the tear film breakup time of rats in the model group was reduced at different treatment times, and there were significant differences compared with the blank control group (p<0.01). Compared with the model group, there were no significant differences in the Hylo group, the 1.5 mg / mL group, the 0.75 mg / mL group, the 0.375 mg / mL group, and the 0.1875 mg / mL group.

[0215] 4.3 Results of tear secretion test

[0216] After successful rat modeling, rats were grouped according to their model scores and drug intervention was initiated, marked as day 0. Tear secretion was examined on days 0, 3, 7, 10, and 14.

[0217] Tear secretion test: After the animal was under general anesthesia, the lower eyelid was pulled down and a 1 mm long folded end of a phenol red cotton thread was placed on the ciliary surface of the lower eyelid near the outer canthus for 20 seconds. The length of the reddened part of the cotton thread was recorded.

[0218] The results are shown in Figure 8 , where ##p < 0.01 vs blank control group, **p < 0.01 vs model group, *p < 0.05 vs model group, aa p < 0.01 vs Heimute group, a p < 0.05 vs Heimute group, p < 0.05 vs 1.5 mg / mL group. From Figure 8 It can be seen that the tear secretion volume of rats in the model group decreased, and there were significant differences compared with the blank control group (p < 0.01). After 14 days of drug administration and treatment in each group, the tear secretion of model rats was lower than that of the blank control group, and there were significant differences in statistical comparison (p < 0.01); after 10 days of drug administration and treatment in each group, the tear secretion in the 0.375 and 0.1875 mg / mL groups was higher than that in the model group, and there were differences in statistical comparison (p < 0.01 or p < 0.05). On the 14th day of treatment, the tear secretion volume in the 0.375 mg / mL new artificial tear group was higher than that in the model group, Heimute group and 1.5 mg / mL group, and there were significant differences in statistical comparison (p < 0.01 or p < 0.05).

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

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

[0221] After the rat models were successfully established, they were grouped according to the model score value and drug intervention was started, recorded as day 0, and H.E. staining examinations were carried out on days 0, 3, 7, 10, and 14 respectively.

[0222] Under light microscopy, the H.E. stained sections of rat corneas were graded, and the pathological grading criteria were as follows:

[0223] Grade 0: The epithelial cell layer is complete, the squamous cell layer is complete, and the stratification is clear. The basal cell layer is complete, arranged neatly and closely adheres to the Bowman's membrane. The basal layer is regular and uniform.

[0224] Grade 1: The epithelial cell layer is complete, the squamous cell layer is reduced, and the stratification is clear. The basal cell layer is complete, arranged neatly and closely adheres to the Bowman's membrane. The basal layer is regular and uniform.

[0225] Grade 2: There is a defect in the epithelial cell layer, the squamous cell layer is reduced, and the stratification is disordered. The basal cell layer is disordered and incomplete, and the arrangement is uneven. The vacuoles in the basal layer increase.

[0226] Grade 3: Epithelial cell layer is missing, squamous cell layer is missing, and stratification is disordered. Basal cell layer is missing, stratified incompletely, and unevenly arranged. Increased basal cell vacuoles.

[0227] Compared with the blank control group, the pathological scores of the model rats were significantly increased (p<0.05). After 14 days of treatment, the pathological scores of the model rats were higher than those of the blank control group (p<0.05); the pathological scores of the Hylo group, the 1.5, 0.75, 0.375, and 0.1875 mg / mL artificial tears groups were lower than those of the model group (p<0.05). Microscopic examination showed that the 0.375 mg / mL group showed the best therapeutic effect compared with the model group, the Hylo group, the 1.5 mg / mL group, and the 0.75 mg / mL group. HE results are shown in Table 12.

[0228] Table 12 Effects of artificial tears on the pathology of a rat model of dry cornea.

[0229]

[0230]

[0231] 4.5 Immunohistochemical results of artificial tears in a rat model of dry cornea

[0232] After successful rat modeling, rats were grouped according to their model scores and drug intervention was initiated, marked as day 0. On day 14, rats were paraffin-embedded, sectioned, and subjected to HE immunohistochemical staining for cytokines (IL-1β, IL-6, IL-17, TNF-α).

[0233] Light microscopy examination scores: Grade 0: No staining of corneal epithelium; Grade 1: Only slight staining in individual areas of corneal epithelium; Grade 2: Staining of the basal cell layer of corneal epithelium, but no staining of the superficial cells; Grade 3: All corneal epithelial cells are stained.

[0234] Studies have shown that the expression of IL-1β, TNF-α, IL-6, and IL-17 by ocular surface epithelial cells plays a crucial role in the inflammatory response of dry eye syndrome. In animal model experiments of dry eye syndrome, the novel artificial tears of this invention significantly reduced the levels of IL-1β, TNF-α, and IL-6 inflammatory factors.

[0235] See results Figure 10 and Figures 11-14 .exist 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 Figure 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 Figure 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 15, where ## p < 0.01 vs blank control group, ** p < 0.01 vs model group, * p < 0.05 vs model group, aa p < 0.01 vs Hylo group, a p < 0.05 vs Hylo group, p < 0.05 vs 1.5 mg / mL group. As Figure 15 It can be seen that compared with the blank control group, the inflammatory indices of the model group were significantly increased (p < 0.01). Compared with the model group, the inflammatory indices of the Hylo group were significantly decreased at 3 and 10 days of use (p < 0.01 or p < 0.05). Compared with the model group, the inflammatory indices of the 0.375 mg / mL artificial tear group were significantly decreased at 3, 7, 10, and 14 days (p < 0.01). Compared with the Hylo group, the inflammatory indices of the 0.375 mg / mL artificial tear group were significantly decreased at 10 and 14 days (p < 0.01 or p < 0.05). Compared with the 1.5 mg / mL group, the inflammatory indices of the 0.375 mg / mL artificial tear group were significantly decreased at 10 and 14 days (p < 0.05).

[0240] It can be seen from the result analysis that after continuous treatment for 10 and 14 days, the inflammatory indices showed a dose-effect trend at concentrations of 1.5, 0.75, 0.375, and 0.1875 mg / mL. As the concentration decreased, the inflammatory index decreased and reached the lowest at 0.375 mg / mL. As the concentration further decreased to 0.1875 mg / mL, the inflammatory index increased again. This indicates that the vicinity of 0.375 mg / mL is the optimal anti-inflammatory concentration.

[0241] The results show that the novel artificial tear of the present invention can reduce the corneal damage of rats with scopolamine-induced keratoconjunctivitis sicca. After instilling the 0.375 mg / mL novel artificial tear for 14 days, the tear secretion volume was higher than that of the model group, and compared with the Hylo sodium hyaluronate eye drop group and other concentration groups, the effect was the best. The novel artificial tear of the present invention can reduce the content of inflammatory factors in the corneal epithelium of the rat scopolamine keratoconjunctivitis sicca model. The contents of the inflammatory factors IL-1β, TNF-α, and IL-6 in the corneal epithelium of the 0.375 mg / mL novel artificial tear group were significantly decreased, and the effect was better than that of the Hylo sodium hyaluronate eye drop group and other concentration groups.

[0242] The novel artificial tear of the present invention can reduce the inflammatory index in the corneal epithelium of the rat scopolamine keratoconjunctivitis sicca model. The effect of reducing the corneal epithelial inflammatory index of the 0.375 mg / mL novel artificial tear group was better than that of the Hylo sodium hyaluronate eye drop group and other concentration groups.

[0243] A comprehensive evaluation of the differences among artificial tear groups showed that the 0.375 mg / mL novel artificial tear demonstrated stable therapeutic effects at all observation time points and across all indicators. After 14 days of continuous treatment, it showed the best results among the groups in terms of fluorescein staining score, tear secretion, corneal epithelial IL1-β, TNF-α, IL-6, and inflammatory index, indicating that it has the best effect in promoting corneal damage repair, increasing tear secretion, and limiting inflammation.

Claims

1. A novel artificial tear fluid containing recombinant human lysozyme, wherein, The novel artificial tears include a main component and excipients. The main component is recombinant human lysozyme. Based on a total weight of 10,000 parts, the novel artificial tears are composed of the following components: 3.75-4 parts of recombinant human lysozyme, 10 parts of sodium hyaluronate, 5.33 parts of anhydrous disodium hydrogen phosphate, 8.62 parts of sodium dihydrogen phosphate monohydrate, 80 parts of sodium chloride, and 9892.05-9892.3 parts of water. The raw material for the recombinant human lysozyme is derived from recombinant human lysozyme stock solution; The recombinant human lysozyme stock solution is a colorless and clear liquid; The sodium chloride content in the recombinant human lysozyme stock solution is 0.36-0.44 w / v%. The recombinant human lysozyme stock solution does not contain disodium hydrogen phosphate or sodium dihydrogen phosphate.

2. The novel artificial tears as claimed in claim 1 wherein, The recombinant human lysozyme has an amino acid sequence that is completely identical to that of natural human lysozyme.

3. The novel artificial tears as claimed in claim 1 wherein, The novel artificial tears have an osmolar concentration of 270-280 mOsmol / kg.

4. The novel artificial tears as claimed in claim 1 wherein, The pH value of the novel artificial tears is 6.4-6.6.

Images