阅读说明：本技术 一种含有重组人溶菌酶的新型人工泪液 (Novel artificial tear containing recombinant human lysozyme ) 是由 史瑾 侯增淼 高恩 李晓颖 杨小琳 赵金礼 于 2018-06-14 设计创作，主要内容包括：本发明提供了一种含有重组人溶菌酶的新型人工泪液。该新型人工泪液包括主成分和辅料,所述主成分为重组人溶菌酶和透明质酸钠,以新型人工泪液的总质量计,重组人溶菌酶和透明质酸钠的含量分别为0.075％-0.300％、0.10％-0.30％。本发明将重组人溶菌酶、透明质酸钠与辅料有效结合,经过眼部外用药物给药,对轻、中度干眼症引起的眼部泪液分泌量下降,眼部干涩,轻微炎症,轻度角结膜损伤等有明显的治疗改善作用。(The invention provides a novel artificial tear containing recombinant human lysozyme. The novel artificial tear comprises a main component and an auxiliary material, wherein the main component comprises recombinant human lysozyme and sodium hyaluronate, and the content of the recombinant human lysozyme and the content of the sodium hyaluronate are respectively 0.075-0.300% and 0.10-0.30% based on the total mass of the novel artificial tear. The invention effectively combines the recombinant human lysozyme, the sodium hyaluronate and the auxiliary materials, and has obvious treatment and improvement effects on eye tear secretion reduction, eye dryness, slight inflammation, mild keratoconjunctival injury and the like caused by light and moderate xerophthalmia through the administration of the eye external medicine.)

1. The novel artificial tear containing the recombinant human lysozyme comprises a main component and an auxiliary material, wherein the main component comprises the recombinant human lysozyme and sodium hyaluronate, and the contents of the recombinant human lysozyme and the sodium hyaluronate are respectively 0.075-0.300% and 0.10-0.30% by mass of the total mass of the novel artificial tear.

2. The novel artificial tear of claim 1 wherein the amino acid sequence of the recombinant human lysozyme is identical to that of human native lysozyme.

3. The novel artificial tear according to claim 1, wherein the recombinant human lysozyme is contained in an amount of 0.150 to 0.300% by mass based on the total mass of the novel artificial tear.

4. The novel artificial tear according to claim 1, wherein the novel artificial tear has a pH of 6.4-6.6, preferably 6.5.

5. The novel artificial tear of claim 1, wherein the excipient comprises a pH stabilizer; preferably, the pH stabilizer comprises sodium citrate, sodium carbonate, sodium bicarbonate, sodium citrate, or a buffer consisting of disodium hydrogen phosphate and sodium dihydrogen phosphate; more preferably, the pH stabilizer is a buffer composed of disodium hydrogen phosphate and sodium dihydrogen phosphate;

preferably, the disodium hydrogen phosphate is disodium hydrogen phosphate dodecahydrate, and the sodium dihydrogen phosphate is sodium dihydrogen phosphate dihydrate, and more preferably, the mass ratio of the two is 20: 9.

6. the novel artificial tear according to claim 1, wherein the osmolality of the novel artificial tear is 285-310 mOsmol/kg.

7. The novel artificial tear of claim 1, 5 or 6, wherein the excipient comprises sodium chloride; preferably, the content of the sodium chloride is 0.70-0.76% of the total mass of the novel artificial tear, and the content of the sodium chloride is calculated by pure solid.

8. The novel artificial tear according to any one of claims 1 to 7, wherein the novel artificial tear comprises, in terms of the total amount of the novel artificial tear being 10000-20000 parts by weight: 7.5-60.0 parts of recombinant human lysozyme, 10.0-60.0 parts of sodium hyaluronate, 20.0-40.0 parts of disodium hydrogen phosphate dodecahydrate, 9.0-18.0 parts of sodium dihydrogen phosphate dihydrate, 70.0-152.0 parts of sodium chloride and 9841.0-19755.0 parts of water for injection.

9. The novel artificial tear according to claim 8, wherein the novel artificial tear comprises, in terms of a total amount of the novel artificial tear being 10000 parts by weight: 15.0-30.0 parts of recombinant human lysozyme, 10.0-30.0 parts of sodium hyaluronate, 20.0 parts of disodium hydrogen phosphate dodecahydrate, 9.0 parts of sodium dihydrogen phosphate dihydrate, 70.0-75.0 parts of sodium chloride and 9841.0-9871.0 parts of water for injection.

10. The novel artificial tear according to claim 8 or 9, wherein the novel artificial tear comprises, in terms of a total amount of the novel artificial tear being 10000 parts by weight: 7.5 parts of recombinant human lysozyme, 10.0 parts of sodium hyaluronate, 20.0 parts of disodium hydrogen phosphate dodecahydrate, 9.0 parts of sodium dihydrogen phosphate dihydrate, 76.0 parts of sodium chloride and 9877.5 parts of water for injection;

or, according to the total amount of the novel artificial tear being 10000 parts by weight, the novel artificial tear comprises: 15.0 parts of recombinant human lysozyme, 10.0 parts of sodium hyaluronate, 20.0 parts of disodium hydrogen phosphate dodecahydrate, 9.0 parts of sodium dihydrogen phosphate dihydrate, 75.0 parts of sodium chloride and 9871.0 parts of water for injection;

or, according to the total amount of the novel artificial tear being 10000 parts by weight, the novel artificial tear comprises: 15.0 parts of recombinant human lysozyme, 30.0 parts of sodium hyaluronate, 20.0 parts of disodium hydrogen phosphate dodecahydrate, 9.0 parts of sodium dihydrogen phosphate dihydrate, 72.0 parts of sodium chloride and 9854.0 parts of water for injection;

or, according to the total amount of the novel artificial tear being 20000 parts by weight, the novel artificial tear comprises: 60.0 parts of recombinant human lysozyme, 20.0 parts of sodium hyaluronate, 40.0 parts of disodium hydrogen phosphate dodecahydrate, 18.0 parts of sodium dihydrogen phosphate dihydrate, 148.0 parts of sodium chloride and 19714.0 parts of water for injection;

or, according to the total amount of the novel artificial tear being 20000 parts by weight, the novel artificial tear comprises: 60.0 parts of recombinant human lysozyme, 60.0 parts of sodium hyaluronate, 40.0 parts of disodium hydrogen phosphate dodecahydrate, 18.0 parts of sodium dihydrogen phosphate dihydrate, 140.0 parts of sodium chloride and 19682.0 parts of water for injection.

Technical Field

The invention relates to a novel artificial tear containing recombinant human lysozyme, belonging to the technical field of medicines.

Background

Symptoms such as eye inflammation, eye dryness, eye fatigue and the like are the most common in daily life of people, and with the increase of work pressure of people, electronic products are updated day by day, computers and mobile phones are used for a longer time, so that the probability of suffering from dry eye and dry eye syndrome is increased continuously. According to statistics, in cities in recent years, dry eye patients are rapidly increasing at a rate of 10% -20% per year, and the incidence rate of people reaches 30% at most. Researches show that people facing a computer screen for more than 9 hours every day have twice of the probability of eye diseases compared with other people; the myopia patients face computers for a long time and are high-risk people with dry eyes; people blink about 20 times in 1 minute under general conditions, when a computer or a mobile phone is used, because of too much attention, the blinking frequency per minute is reduced to 6 times, the focal length is continuously adjusted to ensure the object to be seen to be clear, and the eyes are used with high intensity for a long time.

Dry eye refers to the general term for a variety of diseases characterized by abnormal quality or quantity of tear fluid or abnormal kinetics, resulting in decreased tear film stability, and associated ocular discomfort and/or ocular surface tissue pathology, from any cause. The degree of dry eye classification can be clinically classified into mild, moderate and severe, although there is no gold standard for classification at home and abroad at present. However, generally speaking, mildness is primarily subjective with symptoms, and examinations are primarily signs examinations, asking for medical history; moderate is subjective with the time of tear film rupture, fluorescein staining of cornea, slight keratoconjunctival epithelial damage; the severe condition is mainly that the tear film rupture time is less than 5 seconds, the tear secretion is seriously reduced, the dyeing and coloring of the fluorescein of the keratoconjunctiva are obvious, the corneal epithelial cells are damaged, and the like.

The natural tear is a transparent liquid secreted by organs such as lacrimal gland of human body, is composed of inorganic salt, polysaccharide, protein, lipid and other substances, has multiple functions of barrier, bacteriostasis, sterilization, immunoregulation and the like, and plays an important role in protecting eyeball, nourishing ocular surface tissue, improving visual function and the like. At present, artificial tear products aiming at tear secretion deficiency type xerophthalmia caused by various reasons in the market mainly supplement liquid and preserve moisture, and the products have the characteristics of single component, hypotonicity and the like.

On the other hand, in the market, chemical bacteriostats are often added into artificial tear products aiming at eye discomfort symptoms such as dry eyes, unsmooth eyes, eye fatigue and the like, and the products have good using effect in a short time, but long-term contact with the bacteriostats can cause irritation symptoms such as allergy, dry eyes and the like, damage the cornea or conjunctiva and prevent the cornea or conjunctiva from healing, and iatrogenic keratitis and the like. Numerous studies have demonstrated that bacteriostatic agents in ophthalmic solutions have cytotoxic effects which can affect cellular function and normal metabolism, cause damage to ocular surface tissues if used for extended periods of time, and reduce ocular tolerance to ophthalmic solutions. Some form intractable drug conjunctivitis and drug-induced dry eye. Some clinical investigations have shown that: the discomfort symptoms such as foreign body sensation, stabbing pain sensation, burning sensation and the like of eyes of a patient who takes the eye drop containing the bacteriostatic agent are 2.5 times higher than those of a patient who takes the eye drop containing no bacteriostatic agent, and the discomfort symptoms such as red eyes and dry eyes of the patient are more than 2 times. Also, after changing to an eye drop without bacteriostatic agents, the symptoms were significantly alleviated or reversed. Another survey showed that the therapeutic compliance of many patients was reduced due to the irritation of the bacteriostatic agent in the eye drops, and the frequency of dropping the eye drops was actively reduced, thereby affecting the clinical therapeutic effect.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a novel artificial tear for treating clinically common comprehensive light and moderate xerophthalmia of a video terminal.

In order to achieve the aim, the invention provides a novel artificial tear containing recombinant human lysozyme, which comprises a main component and auxiliary materials, wherein the main component comprises recombinant human lysozyme and sodium hyaluronate, and the contents of the recombinant human lysozyme and the sodium hyaluronate are respectively 0.075-0.300% and 0.10-0.30% by the total mass of the novel artificial tear.

According to a specific embodiment of the present invention, preferably, in the novel artificial tear, the amino acid sequences of the recombinant human lysozyme used are identical to those of the human natural lysozyme. Although the lysozyme has been reported to be used for eye medicaments at present, the lysozyme at present is mostly egg white extracted lysozyme, is chicken lysozyme, has difference from human lysozyme in amino acid composition, is heterologous relative to human body, and often causes side effects such as drug resistance, immunoreaction, anaphylactic reaction and the like when being used for human body. The recombinant human lysozyme (rhLYZ) adopted by the invention can be obtained by a microbial fermentation method, preferably is obtained by fermentation and purification of Pichia pastoris engineering bacteria expressing the human lysozyme, the characteristics are white freeze-dried powder, the molecular weight is 14700D, the purity is more than 98%, the obtained recombinant human lysozyme amino acid sequence is 100% identical to the natural human lysozyme amino acid sequence, and the amino acid sequence is homologous to a human body. The amino acid sequence is as follows (SEQ ID NO: 1):

according to a specific embodiment of the present invention, preferably, in the above novel artificial tear, the content of the recombinant human lysozyme is 0.150% to 0.300% based on the total mass of the artificial tear.

According to a particular embodiment of the present invention, preferably, the pH of the novel artificial tear is 6.4 to 6.6, more preferably 6.5.

According to a specific embodiment of the present invention, preferably, in the above novel artificial tear, the adjuvant comprises a pH stabilizer; more preferably, the pH stabilizer comprises sodium citrate, sodium carbonate, sodium bicarbonate, sodium citrate, or a buffer consisting of disodium hydrogen phosphate and sodium dihydrogen phosphate. When the pH stabilizer is a buffer solution consisting of disodium hydrogen phosphate and sodium dihydrogen phosphate, the pH value of the artificial tear can be stabilized at 6.5, the optimal antibacterial effect and the stability of the formula of the artificial tear are ensured, and the pH value of the artificial tear is closer to that of a natural tear and has no stimulation to eyes. Preferably, the disodium hydrogen phosphate is disodium hydrogen phosphate dodecahydrate, and the sodium dihydrogen phosphate is sodium dihydrogen phosphate dihydrate, and more preferably, the mass ratio of the two is 20: 9.

according to the specific embodiment of the present invention, preferably, the novel artificial tear has an osmolality of 285-310mOsmol/kg, and is isotonic with human tears. The hypertonic solution can make the cornea lose water in the eyes and aggravate the dry eye symptom, the hypotonic solution can make the corneal tissue cells swell and even break, and the isotonic solution system can effectively protect the normal physiological state of the ocular tissue cells and relieve the dry eye symptom.

According to a specific embodiment of the present invention, preferably, in the above novel artificial tear, the adjuvant further comprises sodium chloride. The content of sodium chloride in the novel artificial tear is preferably controlled to be 0.70-0.76% (the content of sodium chloride is calculated by pure solid), so that the recombinant human lysozyme has good solubility, activity and stability in the artificial tear, and the osmotic pressure molar concentration of the artificial tear is 285-310mOsmol/kg, and the recombinant human lysozyme is isotonic with the human tear.

According to an embodiment of the present invention, preferably, the novel artificial tear comprises, in terms of the total amount of the novel artificial tear being 10000-: 7.5-60.0 parts of recombinant human lysozyme, 10.0-60.0 parts of sodium hyaluronate, 20.0-40.0 parts of disodium hydrogen phosphate dodecahydrate, 9.0-18.0 parts of sodium dihydrogen phosphate dihydrate, 70.0-152.0 parts of sodium chloride and 9841.0-19755.0 parts of water for injection. More preferably, the novel artificial tear comprises, in terms of a total amount of the novel artificial tear being 10000 parts by weight: 15.0-30.0 parts of recombinant human lysozyme, 10.0-30.0 parts of sodium hyaluronate, 20.0 parts of disodium hydrogen phosphate dodecahydrate, 9.0 parts of sodium dihydrogen phosphate dihydrate, 70.0-75.0 parts of sodium chloride and 9841.0-9871.0 parts of water for injection.

According to a particular embodiment of the present invention, preferably, the novel artificial tear may have the following particular composition:

according to the total amount of the novel artificial tears being 10000 parts by weight, the novel artificial tears comprise: 7.5 parts of recombinant human lysozyme, 10.0 parts of sodium hyaluronate, 20.0 parts of disodium hydrogen phosphate dodecahydrate, 9.0 parts of sodium dihydrogen phosphate dihydrate, 76.0 parts of sodium chloride and 9877.5 parts of water for injection;

or, the total amount of the novel artificial tears is 10000 parts by weight, and the novel artificial tears comprise: 15.0 parts of recombinant human lysozyme, 10.0 parts of sodium hyaluronate, 20.0 parts of disodium hydrogen phosphate dodecahydrate, 9.0 parts of sodium dihydrogen phosphate dihydrate, 75.0 parts of sodium chloride and 9871.0 parts of water for injection;

or, the total amount of the novel artificial tears is 10000 parts by weight, and the novel artificial tears comprise: 15.0 parts of recombinant human lysozyme, 30.0 parts of sodium hyaluronate, 20.0 parts of disodium hydrogen phosphate dodecahydrate, 9.0 parts of sodium dihydrogen phosphate dihydrate, 72.0 parts of sodium chloride and 9854.0 parts of water for injection;

or, according to the total amount of the novel artificial tears being 20000 parts by weight, the novel artificial tears comprise: 60.0 parts of recombinant human lysozyme, 20.0 parts of sodium hyaluronate, 40.0 parts of disodium hydrogen phosphate dodecahydrate, 18.0 parts of sodium dihydrogen phosphate dihydrate, 148.0 parts of sodium chloride and 19714.0 parts of water for injection;

or, according to the total amount of the novel artificial tears being 20000 parts by weight, the novel artificial tears comprise: 60.0 parts of recombinant human lysozyme, 60.0 parts of sodium hyaluronate, 40.0 parts of disodium hydrogen phosphate dodecahydrate, 18.0 parts of sodium dihydrogen phosphate dihydrate, 140.0 parts of sodium chloride and 19682.0 parts of water for injection.

Aiming at the problems that the number of hits is reduced and the evaporation of tears is too strong due to the fact that a large number of electronic products such as computers, mobile phones and the like are used in the working and living of people in recent years, so that the clinically common comprehensive light and moderate xerophthalmia of the video terminal is caused, the novel artificial tears provided by the invention have the excellent effects of enhancing the secretion quantity of the tears, improving the secretion property of the tears, relieving xerophthalmia symptoms and promoting the repair of cornea damaged cells, and are mainly applied to the treatment of the clinically common comprehensive light and moderate xerophthalmia of the video terminal. The novel artificial tear is sterile, does not contain any chemical bacteriostatic agent, is preservative-free, can be isotonic with human tears, has the functions of maintaining the physiological environment of ocular surfaces and inhibiting bacteria, is more similar to the natural tears of human bodies, can be packaged by daily dosage, can be used up within 24 hours by one branch, and is safe and effective to use.

The novel artificial tear provided by the invention can be prepared by a preparation method comprising the following steps:

(1) adding sodium hyaluronate into water for injection to completely dissolve the sodium hyaluronate to obtain a sodium hyaluronate solution;

(2) adding disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and sodium chloride into the sodium hyaluronate solution to completely dissolve the disodium hydrogen phosphate dodecahydrate, the sodium dihydrogen phosphate dihydrate and the sodium chloride to obtain an auxiliary material solution;

(3) adding the recombinant human lysozyme freeze-dried powder into the auxiliary material solution to completely dissolve the recombinant human lysozyme freeze-dried powder, and then adjusting the pH value of the solution to 6.5 +/-0.1 to obtain a novel artificial tear;

(4) filtering and sterilizing the artificial tears.

The preparation method can be carried out according to the following specific operation steps:

(1) preparation of sodium hyaluronate solution

Sodium hyaluronate is added to water for injection in a ratio and stirred (for example, for about 3 hours) to completely dissolve the sodium hyaluronate, thereby obtaining a sodium hyaluronate solution.

(2) Preparation of the adjuvant solution

Adding disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and sodium chloride into the sodium hyaluronate solution according to the proportion, and stirring until the disodium hydrogen phosphate dodecahydrate, the sodium dihydrogen phosphate dihydrate and the sodium chloride are completely dissolved to obtain an auxiliary material solution.

(3) Preparation of Artificial tears

Adding the recombinant human lysozyme freeze-dried powder into the adjuvant solution according to the proportion, stirring until the recombinant human lysozyme freeze-dried powder is completely dissolved, and then adjusting the pH value of the solution to 6.5 +/-0.1 (for example, adjusting the pH value of the solution by using hydrochloric acid with the concentration of 5% (W/W)). The molar concentration of the osmotic pressure of the artificial tears which is uniformly stirred is detected by adopting a freezing point osmometer and is in the range of 285-310 mOsmol/kg.

(4) Filtration sterilization

The artificial tear is sterilized by a filtration sterilization method, preferably, a 0.22 μm sterilizing filter is used for the filtration sterilization, and the filtration operation pressure for the filtration sterilization can be controlled to be 0.35-0.40 MPa. After filtration sterilization, the sterile artificial tears are transferred to a sterilized material storage tank.

(5) Filling

Filling by adopting a three-in-one aseptic filling technology of blowing, filling and sealing. The blowing-filling-sealing three-in-one technology (BFS) refers to the following steps: in a sterile eye drop production workshop, after plastic particles are extruded and hot-melted in an injection molding machine (170-; bottle body blow molding, liquid medicine filling and preparation sealing are all completed in a set of continuous aseptic production procedures.

In each step, all pipelines for material transportation must be sterilized in advance.

At present, most of common artificial tears are packaged in large dose, are repeatedly opened in the using process and are easy to cause secondary pollution of pathogenic microorganisms such as bacteria and the like after long-time use, so that related chemical bacteriostatic agents are added to ensure the product quality in the using process. A large amount of clinical data and research data show that adverse reactions generated in the ophthalmic treatment process are often caused by bacteriostatic agents, and researches prove that the bacteriostatic agents can indeed cause symptoms such as corneal epithelial cell injury, allergy, eye dryness and the like. In order to ensure the safety and the practicability of the artificial tear in the using process, the artificial tear can be filled into daily dose packages, for example, 0.8 mL/piece, the artificial tear can be used after being opened when used, and a single piece of artificial tear is used up within 24 hours, so that the artificial tear is not easy to cause secondary pollution in the using process through the daily dose packages.

The artificial tear is a local external sterile preparation, the recombinant human lysozyme is active protein, a terminal filtration method is selected for sterilization after raw and auxiliary materials are prepared, sterile liquid is filled aseptically, the activity of the recombinant human lysozyme can be ensured while the artificial tear is ensured to be the sterile preparation, and the inactivation of the lysozyme easily caused by a high-temperature sterilization method is effectively avoided.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the artificial tear of the invention adopts the recombinant human lysozyme and the sodium hyaluronate as main components, does not contain any chemical bacteriostatic agent, does not cause any damage to eyes after long-term use, has no anaphylactic reaction, and is safe and effective.

The invention effectively combines the recombinant human lysozyme, the sodium hyaluronate and the auxiliary materials, and has obvious treatment and improvement effects on eye tear secretion reduction, eye dryness, slight inflammation, mild keratoconjunctival injury and the like caused by light and moderate xerophthalmia through the administration of the eye external medicine.

Drawings

Fig. 1a to 1d show the bacteriostatic effect of the diffusion method of artificial tear paper according to the present invention, wherein fig. 1a is a bacteriostatic map against staphylococcus aureus, fig. 1b is a bacteriostatic map against micrococcus luteus, fig. 1c is a bacteriostatic map against bacillus subtilis, and fig. 1d is a bacteriostatic map against escherichia coli.

FIG. 2 shows a New Zealand rabbit surgical eye tear fern-like crystallization test.

FIG. 3 shows the result of staining corneal epithelium with fluorescein sodium in a New Zealand rabbit operation.

FIG. 4 shows various groups of tear fern-like crystalline forms of New Zealand rabbits administered for 1 week.

FIG. 5 shows the fern-like crystalline form of tear for each group of 2 weeks after administration to New Zealand rabbits.

FIG. 6 shows the fern-like crystalline form of tear for each group of 3 weeks after administration to New Zealand rabbits.

FIG. 7 shows various groups of tear fern-like crystalline forms of New Zealand rabbits administered for 4 weeks.

FIG. 8 shows the results of staining of groups of corneal epithelial fluorescein on New Zealand rabbits at 1 week.

FIG. 9 shows the results of staining of groups of corneal epithelial fluorescein for 2 weeks in New Zealand rabbits.

FIG. 10 shows the results of staining of groups of corneal epithelial fluorescein for 3 weeks in New Zealand rabbits.

FIG. 11 shows the results of the 4-week groups of corneal epithelial fluorescein sodium staining of New Zealand rabbits.

FIG. 12 shows histological micrographs of corneal epithelium (HE staining, 200X) of groups of 4 weeks after administration of New Zealand rabbits.

Detailed Description

The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.

The artificial tear of the invention is obtained by the inventor through deep theory, experimental study and creative work, although the artificial tear can not be completely identical to the natural tear of human body, the artificial tear has the basic characteristics of the tear, including maintaining the physiological environment of the ocular surface (moisturizing, moistening the eyeball, isotonic and stabilizing), bacteriostasis function (the bacteriostasis test of the embodiment 11 shows that the artificial tear has bactericidal effect on gram-positive bacteria), nature (asepsis and no chemical bacteriostat) and the like, and can fulfill the basic functions of the natural tear; the selection and the proportion of the components in the artificial tear formula also have good synergistic effect of mutual promotion.

Firstly, recombinant human lysozyme, one of the main components of artificial tears, is a mucopolysaccharidosis lytic enzyme, is a non-specific immune factor existing in normal body fluids and tissues of human bodies, is an important component of the immune defense system of human eyes, and has certain antibacterial and anti-inflammatory effects. The exogenetic human lysozyme is added dropwise, so that eyes can be better protected, and bacterial infection and inflammation can be prevented. In addition, the human lysozyme is an inherent substance in natural tears of human bodies, and can not generate any toxic or side effect on human bodies after long-term use. Pharmacodynamic tests of a dry eye model in which the artificial tears provided by the embodiment 13 are excessively strong in evaporation to rabbit eyes and accompanied with corneal epithelial cell damage show that the curative effect of the artificial tears is obviously superior to that of commercial artificial tears containing no recombinant human lysozyme and only containing hyaluronic acid single component, and the recombinant human lysozyme with the best effect is contained in the artificial tears in an amount of 0.150-0.300% by weight.

And secondly, sodium hyaluronate, which is the second main component of the artificial tears, is a non-Newtonian fluid, has good biocompatibility, and overcomes the defect that eyelids are not easy to blink while the viscosity of the medicine is increased. In addition, the sodium hyaluronate has good moisturizing and lubricating effects, and can moisten, refresh and feel comfortable to eyes of a patient with xerophthalmia. Normally, hyaluronic acid naturally present on the ocular surface forms a membrane covering the surface of the corneal epithelium, which has specific binding sites for hyaluronic acid. The sodium hyaluronate contained in the artificial tear can form a film to cover the surface of corneal epithelium, so that the recombinant human lysozyme can be remained on the ocular surface for a long time to play a role in inhibiting bacteria, reducing the invasion of external pathogenic microorganisms on corneal epithelial cells and promoting the self-repair of damaged cells. As proved by pharmacodynamic tests of a dry eye model with over-strong eye evaporation and corneal epithelial cell injury of rabbits in the embodiment 13, the artificial tear of the invention has obviously better curative effect than a control solution which does not contain hyaluronic acid and only contains a single component of recombinant human lysozyme.

Thirdly, the inventor surprisingly discovers that the activity and the stability of the recombinant human lysozyme have a certain proportional relationship with the content of sodium chloride in the solution, the higher the content of the recombinant human lysozyme in the solution is, the higher the proportion of the sodium chloride required for complete dissolution is, and the research and test on the solubility of the recombinant human lysozyme and the content of the sodium chloride in the solution in the embodiment 3 prove that when the weight content of the recombinant human lysozyme is 0.300%, the dissolution stability of the recombinant human lysozyme is reduced along with the reduction of the salt concentration in the sodium chloride solution below 0.6%, the recombinant human lysozyme is in a suspension state, the corresponding content and the activity of the recombinant human lysozyme are in a descending trend, and the dissolution stability and the activity of the recombinant human lysozyme in the sodium chloride solution of 0.6% to 6% are basically kept constant; the weight content of sodium chloride in the artificial tear can be controlled to be 0.70-0.76%, and the recombinant human lysozyme can be ensured to have good solubility, activity and stability in the artificial tear, and the osmotic pressure molar concentration of the artificial tear is 285-310mOsmol/kg, and the recombinant human lysozyme is isotonic with the human tear.

In addition, pH is a very important technical control index in artificial tears, relating to the stability, effectiveness and ocular irritation of ophthalmic formulations. The isoelectric point of the active component recombinant human lysozyme is 9.24, and the recombinant human lysozyme has bacteriostatic activity between pH5.0 and 8.0, and the research test on the bacteriostatic activity and the pH of the recombinant human lysozyme given in the example 4 shows that the pH of the optimal bacteriostatic activity is 6.5, the selected disodium hydrogen phosphate-sodium dihydrogen phosphate mixture can stabilize the pH of the artificial tear to be 6.5 +/-0.1, so that the optimal activity condition of the recombinant human lysozyme is ensured, the difference of the pH from the isoelectric point of the recombinant human lysozyme to be 9.24 is more than 2, the long-term stability of the artificial tear is ensured, and meanwhile, the artificial tear with the pH has no stimulation to human eyes.

In conclusion, the good proportion and mutual synergistic effect of the components in the artificial tear of the invention ensure the long-term stability of the artificial tear as a whole (the stability test result given in example 12 shows that the artificial tear is stored for a long time below 25 ℃, the stability is good), the optimal activity of the recombinant human lysozyme improves the bioavailability on the ocular surface, stabilizes the physiological environment on the ocular surface, protects the corneal cells and promotes the repair of damaged cells; effectively treat and relieve eye tear secretion reduction, eye dryness, slight inflammation and the like caused by mild and moderate xerophthalmia. Pharmacodynamic tests of a dry eye model with over-strong eye evaporation and corneal epithelial cell damage of rabbits, which are provided by the embodiment 13, show that compared with the existing products in the market, the artificial tears of the invention have the excellent effects of definitely, effectively and more effectively enhancing the number of tears secreted, improving the nature of tears secreted and promoting the restoration of corneal damaged cells.