阅读说明：本技术 含有重组人溶菌酶和重组人表皮生长因子的新型人工泪液 (Novel artificial tears containing recombinant human lysozyme and recombinant human epidermal growth factor ) 是由 史瑾 侯增淼 高恩 李晓颖 庞荣荣 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和重组人表皮生长因子的新型人工泪液。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、重组人表皮生长因子和透明质酸钠,以新型人工泪液的总质量计,重组人溶菌酶、重组人表皮生长因子和透明质酸钠的含量分别为0.075％-0.300％、0.002％-0.004％、0.10％-0.30％。本发明将重组人溶菌酶、重组人表皮生长因子、透明质酸钠与辅料有效结合,经过眼部外用药物给药,对中、重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 and recombinant human epidermal growth factor. The novel artificial tear comprises a main component and an auxiliary material, wherein the main component comprises recombinant human lysozyme, recombinant human epidermal growth factor and sodium hyaluronate, and the contents of the recombinant human lysozyme, the recombinant human epidermal growth factor and the sodium hyaluronate are respectively 0.075-0.300%, 0.002-0.004% and 0.10-0.30% by total mass of the novel artificial tear. The invention effectively combines the recombinant human lysozyme, the recombinant human epidermal growth factor, the sodium hyaluronate and the auxiliary materials, and has obvious treatment and improvement effects on the reduction of eye tear secretion, eye dryness, slight inflammation, mild keratoconjunctival injury and the like caused by moderate and severe xerophthalmia through the administration of the externally-applied eye medicine.)

1. The novel artificial tear containing the recombinant human lysozyme and the recombinant human epidermal growth factor comprises a main component and auxiliary materials, wherein the main component comprises the recombinant human lysozyme, the recombinant human epidermal growth factor and sodium hyaluronate, and the contents of the recombinant human lysozyme, the recombinant human epidermal growth factor and the sodium hyaluronate are respectively 0.075-0.300%, 0.002-0.004% and 0.10-0.30% by 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 a human natural lysozyme; the amino acid sequences of the recombinant human epidermal growth factor and the natural human epidermal growth factor are completely consistent.

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; more 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, 0.2-0.8 part of recombinant human epidermal growth factor, 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 9840.6-19754.6 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, 0.2-0.4 part of recombinant human epidermal growth factor, 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 9840.6-9870.8 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, 0.2 part of recombinant human epidermal growth factor, 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 9877.3 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, 0.2 part of recombinant human epidermal growth factor, 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 9870.8 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, 0.2 part of recombinant human epidermal growth factor, 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 9853.8 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, 0.4 part of recombinant human epidermal growth factor, 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 19713.6 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, 0.8 part of recombinant human epidermal growth factor, 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 19681.2 parts of water for injection.

Technical Field

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

Background

Eye inflammation, eye dryness, eye fatigue and other symptoms are most common in daily life of people, and along with the increase of working pressure of people, electronic products are updated day by day and computers and mobile phones are used for a longer time; the chances of developing dry eye and dry eye syndrome are increasing. 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 of human body is a transparent liquid secreted by organs of human body, such as lacrimal gland, etc., and is formed from several substances of inorganic salt, polysaccharide, protein and lipid, etc., and possesses several functions of protecting eyeball, nourishing ocular surface tissue and improving visual function, etc. 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 moderate and severe xerophthalmia of a video terminal.

In order to achieve the purpose, the invention provides a novel artificial tear containing recombinant human lysozyme and recombinant human epidermal growth factor, which comprises a main component and auxiliary materials, wherein the main component comprises recombinant human lysozyme, recombinant human epidermal growth factor and sodium hyaluronate, and the contents of the recombinant human lysozyme, the recombinant human epidermal growth factor and the sodium hyaluronate are respectively 0.075-0.300%, 0.002-0.004% and 0.10-0.30% by 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 is obtained by a microbial fermentation method, in particular to a recombinant human lysozyme (rhLYZ) which is obtained by fermenting and purifying pichia pastoris engineering bacteria expressing human lysozyme, the characteristics of the recombinant human lysozyme 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 recombinant human lysozyme is homologous to a human body. The amino acid sequence is as follows (SEQ ID NO: 1):

1KVFERCELAR TLKRLGMDGY RGISLANWMC LAKWESGYNT RATNYNAGDR

51STDYGIFQIN SRYWCNDGKT PGAVNACHLS CSALLQDNIA DAVACAKRVV

101RDPQGIRAWV AWRNRCQNRD VRQYVQGCGV

the recombinant human epidermal growth factor (rhEGF) adopted by the invention is obtained by a microbial fermentation method, and specifically is obtained by fermenting and purifying Pichia pastoris engineering bacteria expressing the human epidermal growth factor, the characteristics are white freeze-dried powder, the purity is more than 95%, the obtained amino acid sequence of the recombinant human epidermal growth factor is 100% identical to the amino acid sequence of a natural human epidermal growth factor, and the amino acid sequence is as follows (SEQ ID NO: 5):

1NSDSECPLSH DGYCLHDGVC MYIEALDKYA CNCVVGYIGE RCQYRDLKWW

51ELR

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. The recombinant human epidermal growth factor has no function of promoting cell proliferation when the cell is endangered to die in a hypertonic or hypotonic environment, and can fully play a role when acting on active healthy cells in an isotonic physiological environment.

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 a specific embodiment of the present invention, preferably, the novel artificial tear includes, in terms of a total amount of 10000-: 7.5-60.0 parts of recombinant human lysozyme, 0.2-0.8 part of recombinant human epidermal growth factor, 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 9840.6-19754.6 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, 0.2-0.4 part of recombinant human epidermal growth factor, 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 9840.6-9870.8 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, 0.2 part of recombinant human epidermal growth factor, 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 9877.3 parts of water for injection;

or, the total amount of the novel artificial tears is 10000 parts by weight, and the artificial tears comprise: 15.0 parts of recombinant human lysozyme, 0.2 part of recombinant human epidermal growth factor, 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 9870.8 parts of water for injection;

or, the total amount of the novel artificial tears is 10000 parts by weight, and the artificial tears comprise: 15.0 parts of recombinant human lysozyme, 0.2 part of recombinant human epidermal growth factor, 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 9853.8 parts of water for injection;

or, the total amount of the novel artificial tears is 20000 parts by weight, and the artificial tears comprise: 60.0 parts of recombinant human lysozyme, 0.4 part of recombinant human epidermal growth factor, 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 19713.6 parts of water for injection;

or, the total amount of the novel artificial tears is 20000 parts by weight, and the artificial tears comprise: 60.0 parts of recombinant human lysozyme, 0.8 part of recombinant human epidermal growth factor, 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 19681.2 parts of water for injection.

Aiming at the clinically common video terminal comprehensive xerophthalmia caused by the fact that the number of times of eyes is reduced and tears are evaporated too strongly due to the fact that a large number of electronic products such as computers, mobile phones and the like are used in working life of people in recent years, 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 and promoting the repair of cornea damaged cells, and are mainly applied to the treatment of clinically common video terminal comprehensive moderate-severe xerophthalmia. 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 and the recombinant human epidermal growth factor freeze-dried powder into the auxiliary material solution to completely dissolve the two, and then adjusting the pH value of the solution to 6.5 +/-0.1 to obtain the artificial tear;

(4) filtering and sterilizing the artificial tears.

Preferably, the preparation method of the present invention further comprises: (5) and filling the artificial tears subjected to filtration sterilization.

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 the above 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 above 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 and the recombinant human epidermal growth factor freeze-dried powder into the adjuvant solution according to the proportion, stirring until the recombinant human lysozyme freeze-dried powder and the recombinant human epidermal growth factor freeze-dried powder are 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 adopting 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 main components of the recombinant human lysozyme and the recombinant human epidermal growth factor are active proteins, a terminal filtration method is selected for sterilization after the raw and auxiliary materials are prepared, sterile liquid is filled aseptically, the activity of the recombinant human lysozyme and the recombinant human epidermal growth factor can be ensured while the eye drops are ensured to be the sterile preparation, and the inactivation of the recombinant human lysozyme and the recombinant human epidermal growth factor 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, the recombinant human epidermal growth factor and the sodium hyaluronate as main components, does not contain any chemical bacteriostatic agent, can 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 recombinant human epidermal growth factor, the sodium hyaluronate and the auxiliary materials, is mainly used for treating moderate and severe xerophthalmia, and has obvious treatment and improvement effects on reduction of eye tear secretion, eye dryness, slight inflammation, corneal injury and the like caused by moderate and severe xerophthalmia through external administration of eye medicines.

Drawings

FIG. 1a shows the result of staining with sodium hyaluronate and fluorescein in corneal epithelium of rat in control group before modeling.

FIG. 1b shows the result of staining with sodium fluorescein in rat corneal epithelium of the recombinant human lysozyme control group before modeling.

FIG. 1c shows the result of staining with rat corneal epithelial fluorescein sodium in the control group of recombinant human epidermal growth factor before modeling.

FIG. 1d shows the results of rat corneal epithelial fluorescein sodium staining in the group of artificial tears prior to molding.

Figure 2a shows the results of sodium hyaluronate corneal epithelium fluorescein sodium staining in rat control group after 10 days of molding.

FIG. 2b shows the result of corneal epithelial fluorescein sodium staining of rats in the recombinant human lysozyme control group 10 days after the molding.

FIG. 2c shows the result of corneal epithelial fluorescein sodium staining of rats in the recombinant human epidermal growth factor control group 10 days after molding.

FIG. 2d shows the results of fluorescein sodium staining of corneal epithelium of rat in artificial tear group after 10 days of molding.

Figure 3a shows the results of sodium hyaluronate corneal epithelium fluorescein sodium staining in rats in the control group 5 days after treatment.

FIG. 3b shows the results of corneal epithelial fluorescein sodium staining of rats in the recombinant human lysozyme control group 5 days after treatment.

FIG. 3c shows the result of corneal epithelial fluorescein sodium staining of rats in the recombinant human epidermal growth factor control group 5 days after the treatment.

Figure 3d shows the results of fluorescein sodium staining of rat corneal epithelium in artificial tear group after 5 days of treatment.

Figure 4a shows the results of HE staining of pathological sections of rat corneal epithelium (400 x) in the sodium hyaluronate control group 5 days after treatment.

FIG. 4b shows the HE staining results (400X) of the corneal epithelial pathological section of rats in the recombinant human lysozyme control group 5 days after treatment.

FIG. 4c shows the HE staining results of pathological sections of rat corneal epithelium in the control group of recombinant human epidermal growth factor (400X) after 5 days of treatment.

FIG. 4d shows the results of HE staining of pathological sections of corneal epithelium of rats in the artificial tear group (400X) after 5 days of treatment.

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 novel artificial tear of the invention can not be completely identical with the natural tear of human body, the novel artificial tear of the invention has the basic characteristics of the tear, including the functions of maintaining the physiological environment of the ocular surface (moisturizing, moistening the eyeball, isotonic and stabilizing), inhibiting bacteria and promoting the proliferation of corneal epithelial cells (the experiment of example 13 shows that the artificial tear has the functions of inhibiting bacteria and promoting the proliferation of cells), being natural (sterile and without chemical bacteriostat), and the like, and can fulfill the basic functions of the natural tear; the selection and the proportion of each component in the novel artificial tear 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.

Another effective active ingredient of the novel artificial tears is recombinant human epidermal growth factor. Human epidermal growth factor is an important growth factor in human endocrine, widely exists in various tissues of human body, and can promote division and growth of epidermal cells. The epidermal growth factor has the function of promoting the growth of cells such as keratinocytes, fibroblasts, smooth muscle cells, endothelial cells and the like, is also an important cytokine for repairing corneal epithelium and stroma injuries, and can effectively repair corneal injuries.

The sodium hyaluronate is a non-Newtonian fluid, has good biocompatibility, and overcomes the defect that eyelids are not easy to blink while increasing the viscosity of the medicine. 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 three main substances are combined in an optimal effect, sodium hyaluronate contained in the artificial tears forms a film to cover the surface of corneal epithelium, so that recombinant human lysozyme and recombinant human epidermal growth factors can be remained on the ocular surface for a long time, the recombinant human lysozyme plays a role in bacteriostasis, the invasion of external pathogenic microorganisms to corneal epithelium is reduced, and the recombinant human epidermal growth factors promote the repair and regeneration of corneal epithelium damaged cells. The pharmacodynamic test of the artificial tear on a benzalkonium chloride-induced rat dry eye with severe corneal injury model shows that the curative effect of the artificial tear is obviously superior to that of a hyaluronic acid reference substance containing only a single component, a recombinant human lysozyme reference substance containing a single component and a commercially available eye drop product containing a single component of epidermal growth factor.

Secondly, when a plurality of active proteins or main components are combined together, the main components may interact with each other to cause the respective efficacy to be reduced, the curative effect of the composition is rather inferior to that of a single component, the three main components of the invention can play excellent combined efficacy, and besides the respective characteristics, the selection and compatibility of auxiliary materials also have important roles.

The inventor surprisingly discovers that the activity and stability of the recombinant human lysozyme have a certain proportional relationship with the content of sodium chloride in a solution in the preparation, purification and test processes of the recombinant human lysozyme, 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 5 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 with the concentration of less than 0.6%, the recombinant human lysozyme is in a suspension state, the corresponding content and 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 with the concentration of 0.6% -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 proportion can simultaneously ensure 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, so that the recombinant human lysozyme has isotonic with the human tear.

pH is a very important technical control index in artificial tears, and is related to the stability, effectiveness and ocular irritation of ophthalmic preparations. 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 of the bacteriostatic activity and the pH of the recombinant human lysozyme in example 6 shows that the pH of the optimal bacteriostatic activity is 6.5; the isoelectric point of the active component recombinant human epidermal growth factor is 4.67, and the cell proliferation promoting function of the recombinant human epidermal growth factor can fully exert the effect by acting on viable and healthy cells in a physiological environment. The selected disodium hydrogen phosphate-sodium dihydrogen phosphate ratio can stabilize the pH of the eye drops to be 6.5 +/-0.1, is basically close to the physiological pH of a human body, ensures the optimal activity conditions of the recombinant human lysozyme and the recombinant human epidermal growth factor, has a pH difference of more than 1.5 from the isoelectric points of the recombinant human lysozyme and the recombinant human epidermal growth factor, ensures the long-term stability of the recombinant human lysozyme and the recombinant human epidermal growth factor, and meanwhile, the artificial tears with the pH have no stimulation to the eyes of the human body.

The selection and the proportion of the three inorganic salts form an auxiliary material solution, and then the auxiliary material solution is combined with the three main components to form the artificial tear, and the compatibility stability research test of the recombinant human lysozyme and the recombinant human epidermal growth factor in the artificial tear of the embodiment 13 is carried out; example 14 forced high frequency vibration test of artificial tears; example 15 Long-term stability and accelerated testing of artificial tears indicate that the formulation is stable; the functions of the components are mutually cooperated, and compared with a single-component control product in pharmacodynamic tests of a rat dry eye with severe corneal injury model induced by benzalkonium chloride in example 16, the artificial tear has the excellent effects of definitely, effectively and more effectively enhancing the tear secretion quantity and promoting the corneal injury cell repair.