WO2019237634A1 - 一种含有重组人溶菌酶的新型人工泪液 - Google Patents
一种含有重组人溶菌酶的新型人工泪液 Download PDFInfo
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- WO2019237634A1 WO2019237634A1 PCT/CN2018/112518 CN2018112518W WO2019237634A1 WO 2019237634 A1 WO2019237634 A1 WO 2019237634A1 CN 2018112518 W CN2018112518 W CN 2018112518W WO 2019237634 A1 WO2019237634 A1 WO 2019237634A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/04—Artificial tears; Irrigation solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/02—Local antiseptics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates to a novel artificial tear solution containing recombinant human lysozyme, and belongs to the technical field of medicine.
- Eye inflammation, eye dryness, and eye fatigue are the most common in people's daily lives. With the increase of people's work pressure, the electronic products are increasingly updated, and the use of computers and mobile phones for more and more time has led to dryness.
- the risk of eye and dry eye syndrome is increasing. According to statistics, in recent years, dry eye patients in cities are rapidly increasing at a rate of 10% -20% per year, and the incidence of the population is up to 30%. Studies have found that people who face computer screens for more than 9 hours a day are twice as likely to suffer from eye diseases than other people; patients with myopia who face computers for a long period of time are more at risk for dry eye conditions; in general, people Blink about 20 times in 1 minute.
- Dry eye disease is a general term for a variety of diseases that cause abnormal tear quality or quantity or kinetic abnormalities caused by any cause, leading to a decrease in tear film stability, accompanied by ocular discomfort and / or ocular surface tissue lesions.
- the classification of dry eye can be divided into mild, moderate and severe clinically, although there is no gold standard for classification at home and abroad.
- Human natural tear fluid is a transparent liquid secreted by organs such as the human lacrimal gland. It is composed of various substances such as inorganic salts, polysaccharides, proteins, lipids, etc. It has a variety of functions such as barrier, bacteriostasis, sterilization, and immune regulation. The nutritional ocular surface organization and the improvement of visual function play an important role.
- artificial tear products for the lack of tear secretion-type dry eye caused by various reasons on the market are mainly supplemented with liquids, mainly moisturizing.
- the products themselves are characterized by a single ingredient and low permeability.
- an object of the present invention is to provide a new type of artificial tear fluid for treating clinically common video terminals with comprehensive light and moderate dry eye.
- the present invention provides a new artificial tear containing recombinant human lysozyme, which comprises a main component and an auxiliary material, the main components are recombinant human lysozyme and sodium hyaluronate, and the total mass of the new artificial tear In total, the contents of recombinant human lysozyme and sodium hyaluronate were 0.075% -0.300% and 0.10% -0.30%, respectively.
- the amino acid sequence of the recombinant human lysozyme and the human natural lysozyme are completely identical.
- the current lysozyme is mostly lysozyme extracted from egg white and chicken lysozyme, which differs from human lysozyme in amino acid composition. It is said to be heterogeneous, and its use in humans often causes side effects such as drug resistance, immune responses, and allergic reactions.
- the recombinant human lysozyme (rhLYZ) used in the present invention can be obtained by microbial fermentation, preferably obtained by fermentation and purification of Pichia pastoris engineering bacteria expressing human lysozyme.
- the trait is a white lyophilized powder with a molecular weight of 14700D.
- the purity is greater than 98%.
- the amino acid sequence of the recombinant human lysozyme obtained is 100% identical to the amino acid sequence of the natural human lysozyme, which is homologous to the human body.
- the amino acid sequence is as follows (SEQ ID NO: 1):
- the content of the recombinant human lysozyme is 0.150% -0.300% based on the total mass of the artificial tear fluid.
- the pH of the novel artificial tear fluid is 6.4-6.6, and more preferably 6.5.
- the auxiliary material includes a pH stabilizer; more preferably, the pH stabilizer includes sodium citrate, sodium carbonate, sodium bicarbonate, and citric acid.
- Sodium or a buffer solution composed of disodium hydrogen phosphate and sodium dihydrogen phosphate When the pH stabilizer is a buffer solution composed of disodium hydrogen phosphate and sodium dihydrogen phosphate, the pH value of artificial tears can be stabilized at 6.5 to ensure the best bacteriostatic effect of artificial tears and the stability of the formula. It is close to the pH of natural tears and has no irritation to the eyes.
- the disodium hydrogen phosphate is disodium hydrogen phosphate dodecahydrate
- the sodium dihydrogen phosphate is sodium dihydrogen phosphate dihydrate, and more preferably, the mass ratio of the two is 20: 9.
- the osmolality of the novel artificial tear is 285-310 mOsmol / kg, which is isotonic with human tear.
- Hypertonic solution can make the cornea lose water in the eye and increase the dryness of the eye.
- Hypotonic solution can make the corneal tissue cells swell or even rupture.
- the isotonic solution system can effectively protect the normal physiological state of the ocular tissue cells. , Reduce the symptoms of dry eyes.
- the auxiliary material further includes sodium chloride.
- the novel artificial tear solution of the present invention preferably controls the content of sodium chloride to 0.70% -0.76% (the content of sodium chloride is based on pure solids), which can simultaneously ensure that the recombinant human lysozyme has good solubility, activity,
- the stability and osmolality of artificial tears are 285-310mOsmol / kg, which is isotonic with human tears.
- the new artificial tears include: 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.
- the new artificial tears include: 15.0-30.0 parts of recombinant human lysozyme, 10.0-30.0 parts of sodium hyaluronate, and 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.
- the novel artificial tear fluid may have the following specific composition:
- the new artificial tears include: 7.5 parts of recombinant human lysozyme, 10.0 parts of sodium hyaluronate, 20.0 parts of disodium hydrogen phosphate dodecahydrate, and 9.0 sodium dihydrogen phosphate dihydrate Parts, 76.0 parts of sodium chloride, 9877.5 parts of water for injection;
- the new artificial tears include: 15.0 parts of recombinant human lysozyme, 10.0 parts of sodium hyaluronate, 20.0 parts of disodium hydrogen phosphate dodecahydrate, and dihydrogen phosphate dihydrate 9.0 parts of sodium, 75.0 parts of sodium chloride, 9871.0 parts of water for injection;
- the new artificial tears include: 15.0 parts of recombinant human lysozyme, 30.0 parts of sodium hyaluronate, 20.0 parts of disodium hydrogen phosphate dodecahydrate, and dihydrogen phosphate dihydrate 9.0 parts of sodium, 72.0 parts of sodium chloride, 9854.0 parts of water for injection;
- the new artificial tears include: 60.0 parts of recombinant human lysozyme, 20.0 parts of sodium hyaluronate, 40.0 parts of disodium hydrogen phosphate dodecahydrate, and dihydrogen phosphate dihydrate 18.0 parts of sodium, 148.0 parts of sodium chloride, 19714.0 parts of water for injection;
- the new artificial tears include: 60.0 parts of recombinant human lysozyme, 60.0 parts of sodium hyaluronate, 40.0 parts of disodium hydrogen phosphate dodecahydrate, and dihydrogen phosphate dihydrate 18.0 parts of sodium, 140.0 parts of sodium chloride, and 19682.0 parts of water for injection.
- the present invention provides The new type of artificial tears has the excellent effect of enhancing the amount of tear secretion, improving the properties of tear secretion, reducing the symptoms of dry eye, and promoting the repair of corneal damaged cells. It is mainly used in the treatment of clinical video terminal comprehensive light and moderate dry eye syndrome.
- the novel artificial tear fluid of the present invention is sterile, does not contain any chemical bacteriostatic agent, has no preservatives, can be isotonic with human tear fluid, has the function of maintaining the physiological environment of the ocular surface and antibacterial, and is closer to the human natural tear fluid. Packaging, single use up within 24 hours, safe and effective use.
- novel artificial tears provided by the present invention may be prepared by a preparation method including the following steps:
- the osmolality of the artificially stirred tear should be in the range of 285-310mOsmol / kg.
- the artificial tear fluid is sterilized by a filtering and sterilizing method.
- a 0.22 ⁇ m sterilizing filter is used for filtering and sterilizing, and the filtering operation pressure used for filtering and sterilizing can be controlled to 0.35-0.40 MPa.
- the sterile artificial tear fluid is transferred to the sterilized material storage tank.
- BFS Blow-Fill-Seal
- artificial tears are often packaged in large doses, which are repeatedly opened during use, and long-term use is likely to cause secondary pollution of pathogenic microorganisms such as bacteria. Therefore, related chemical bacteriostatic agents are added to ensure product quality during use.
- a large amount of clinical data and research data show that the adverse reactions during ophthalmic treatment are often caused by bacteriostatic agents.
- bacteriostatic agents can indeed cause corneal epithelial cell damage, allergies and dry eyes.
- it can be filled into a daily dose package, for example, 0.8mL / bran, which is used immediately when used, and used up within 24 hours of a single dose. Packaging makes artificial tears less likely to cause secondary contamination during use.
- the artificial tear solution of the present invention is a topical sterile preparation, and the recombinant human lysozyme is an active protein.
- the terminal filtration method is used for sterilization, and the sterile solution is aseptically filled.
- the activity of the recombinant human lysozyme can be ensured, and the lysozyme inactivation easily caused by the high-temperature sterilization method can be effectively avoided.
- the artificial tear fluid of the present invention uses recombinant human lysozyme and sodium hyaluronate as main components, does not contain any chemical bacteriostatic agent, and does not cause any damage to the eyes for a long time, has no allergic reaction, and is safe and effective.
- the present invention effectively combines recombinant human lysozyme, sodium hyaluronate and excipients, and through topical drug administration to the eye, it reduces ocular tear secretion caused by mild to moderate dry eye disease, dry eyes, slight inflammation, and mild Degree of corneal and conjunctival injury has obvious therapeutic improvement.
- FIG. 1a to 1d show the bacteriostatic effect of the artificial tear paper sheet diffusion method of the present invention, wherein FIG. 1a is a bacteriostatic chart for Staphylococcus aureus, and FIG. 1b is a bacteriostatic chart for Micrococcus luteus, FIG. 1c is a bacteriostatic map against Bacillus subtilis, and FIG. 1d is a bacteriostatic map against Escherichia coli.
- Figure 2 shows a fern-like crystal test of New Zealand rabbit surgical tears.
- Figure 3 shows the results of sodium fluorescein staining of corneal epithelium in New Zealand rabbit surgical eyes.
- Figure 4 shows the tear-shaped fern-like crystal morphology of each group of New Zealand rabbits for 1 week.
- Figure 5 shows the tear-shaped fern-like crystal morphology of each group of New Zealand rabbits for 2 weeks.
- Figure 6 shows the tear-shaped fern-like crystal morphology of each group of New Zealand rabbits for 3 weeks.
- Figure 7 shows the tear-shaped fern-like crystal morphology of each group of New Zealand rabbits for 4 weeks.
- Figure 8 shows the results of sodium fluorescein staining of corneal epithelium in each group for one week of New Zealand rabbit medication.
- Figure 9 shows the results of corneal epithelial sodium staining in each group of New Zealand rabbits for 2 weeks.
- FIG. 10 shows the results of sodium fluorescein staining of corneal epithelium in each group of 3 weeks of New Zealand rabbit medication.
- Fig. 11 shows the results of sodium fluorescein staining of corneal epithelium in each group of 4 weeks of New Zealand rabbit medication.
- Figure 12 shows histological micrographs of corneal epithelium (HE staining, 200 ⁇ ) in each group of New Zealand rabbits administered for 4 weeks.
- the artificial tears of the present invention are obtained by the inventors through in-depth theory, experimental research, and creative work. Although the artificial tears are not completely equivalent to human natural tears, they already have the basic characteristics of tears, including maintaining the physiological environment of the ocular surface ( Moisturizing, moisturizing eyeballs, isotonic, stable), bacteriostatic function (Example 11 bacteriostatic test showed bactericidal effect on Gram-positive bacteria), natural (sterile, no chemical bacteriostatic agent), etc., can perform natural tears The basic function of the composition; the selection and proportion of the components in the artificial tears formula also have a good synergistic effect.
- human lysozyme which is one of the main components of artificial tears, is a mucopolysaccharide lyase, which is a non-specific immune factor that exists in normal human body fluids and tissues, and is an important part of the human eye's immune defense system. , Has a certain antibacterial and anti-inflammatory effect. Exogenous drop of human lysozyme can better protect the eyes and prevent the occurrence of bacterial infection and inflammation.
- human lysozyme is an inherent substance in the human body's natural tear fluid, and it will not cause any toxic or side effects to the human body for a long time.
- Example 13 The dry eye model pharmacodynamics test of artificial tears in Example 13 with excessive evaporation on rabbit eyes with corneal epithelial cell damage showed that the efficacy of the artificial tears of the present invention is significantly better than that without recombinant human lysozyme.
- a commercially available artificial tear product containing a single component of hyaluronic acid, and the recombinant human lysozyme that also exhibits the best effect has a weight content of 0.150% -0.300%.
- sodium hyaluronate which is the second main component of artificial tears, is a non-Newtonian fluid, which has good biocompatibility, and overcomes the shortcomings of eyelids that are not easy to blink while increasing the viscosity of the drug.
- sodium hyaluronate has good moisturizing and lubricating effects, and can make the eyes of patients with dry eye feel moist and refreshing and comfortable.
- the hyaluronic acid naturally present on the ocular surface forms a film covering the surface of the corneal epithelium, and the corneal epithelium has a specific binding site with hyaluronic acid.
- the sodium hyaluronate contained in the artificial tear fluid of the present invention will form a film covering the surface of the corneal epithelium, so that the recombinant human lysozyme can remain on the ocular surface for a long time, exert a bacteriostatic effect, and reduce the effect of external pathogenic microorganisms on the corneal epithelial cells. Invasion promotes self-repair of damaged cells.
- the efficacy of the artificial tear solution of the present invention is significantly better than that without hyaluronic acid and only contains recombinant human lysobacteria. Enzyme single component reference solution.
- the inventors surprisingly discovered during the preparation, purification and testing of recombinant human lysozyme that the activity and stability of recombinant human lysozyme has a certain proportional relationship with the sodium chloride content in the solution.
- the recombinant human lysozyme is in solution. The higher the content, the higher the proportion of sodium chloride required for complete dissolution. According to the research test of the solubility of the recombinant human lysozyme and the sodium chloride content in the solution in Example 3, the weight of the recombinant human lysozyme was 0.300%.
- the solubility stability of recombinant human lysozyme decreases and becomes suspended, and the corresponding content and activity show a downward trend, ranging from 0.6% to 6%.
- the solubility stability and activity of the recombinant human lysozyme in the sodium chloride solution remain substantially constant; the weight content of sodium chloride in the artificial tear solution of the present invention can be controlled to 0.70% -0.76%, which can simultaneously ensure that the recombinant human lysozyme in the artificial tear solution It has good solubility, activity, stability and osmolality of artificial tears from 285-310mOsmol / kg, and isotonic with human tears.
- pH is a very important technical control index in artificial tears, which is related to the stability, effectiveness and eye irritation of ophthalmic preparations.
- the active ingredient of recombinant human lysozyme has an isoelectric point of 9.24, and has a bacteriostatic activity between pH 5.0 and 8.0.
- the research test of the bacteriostatic activity and pH of the recombinant human lysozyme given in Example 4 shows its best bacteriostatic activity.
- the pH is 6.5, and the ratio of disodium hydrogen phosphate-sodium dihydrogen phosphate used in the present invention can stabilize the artificial tears at a pH of 6.5 ⁇ 0.1, ensuring the optimal activity conditions of recombinant human lysozyme, and isoelectricity from the recombinant human lysozyme.
- Point 9.24 differs by more than 2 pHs, ensuring long-term stability of artificial tears, and at the same time, artificial tears at this pH have no irritation to the human eye.
- the good ratio and synergy of the components in the artificial tear of the present 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 Long-term storage below 25 ° C, good stability), the best activity of recombinant human lysozyme, improving its bioavailability on the ocular surface, stabilizing the physiological environment of the ocular surface, protecting corneal cells, and promoting repair of damaged cells ; Effective treatment to alleviate the decrease of ocular tear secretion caused by mild and moderate dry eye, dry eyes, slight inflammation and so on.
- the pharmacodynamic test of the dry eye model with excessive evaporation of the rabbit eye and corneal epithelial cell damage given in Example 13 shows that the artificial tear of the present invention has a clear and effective effect compared with the existing products on the market. More effectively enhance the amount of tear secretion, improve the properties of tear secretion, and promote the excellent effect of corneal damage cell repair.
- the human skin cDNA was used as a template, and the designed LYZ-XU and LYZ-EL were used as primers. PCR was performed at an annealing temperature of 56 ° C. The PCR results showed specific bands at the expected positions. Recycling, then Xho I (purchased from Thermo Fisher) and EcoR I (purchased from Thermo Fisher) were double-digested with the pPIC9K vector (purchased from Invitrogen), and the digested product was recovered and ligated with DNA The enzyme was ligated and transformed into E. coli, and the plasmid was extracted and identified by sequencing. A human lysozyme expression vector was obtained and named pPIC9K-LYZ.
- the colonies grown on the MD culture plate were inoculated with a sterile toothpick into YPD (yeast extract (Yeast extract) 1 wt%, G418, 1g / L, 2g / L, 3g / L, 4g / L).
- YPD yeast extract
- Polypepton (2wt%, Dexrose 2wt%) plates were cultured at 30 ° C, and transformants were obtained by shake flask screening.
- the selected transformants were inoculated into 4 BMGY (1% yeast extract), 2% peptone, 100mmol / L potassium phosphate solution, pH 6.0, 1.34% YNB (yeast) 1% glycerol) culture medium in a conical flask, in a constant temperature culture shaker, cultured at 29 ° C and 225 revolutions for 60-70 hours, and then connected to the seed tank.
- BMGY 1% yeast extract
- peptone 100mmol / L potassium phosphate solution
- pH 6.0 pH 6.0
- YNB 1% glycerol
- the first-stage seeds were transferred to a medium containing 3 L of FBS (1 L containing glycerol 40 g, K 2 SO 4 18.2 g, H 3 PO 4 26.7 mL, CaSO 4 ⁇ 2H 2 O 0.93 g, MgSO 4 14.9 g, KOH 4.13
- FBS glycerol
- the temperature of the tank is controlled at 29.0 ⁇ 1.0 ° C.
- the tank pressure is 0.050 ⁇ 0.010 MPa
- the pH is 5.0.
- aeration and stirring speed are adjusted to maintain dissolved oxygen at about 30%.
- After 16 hours of secondary seed culture the dissolved oxygen was observed, and the dissolved oxygen increased significantly (10% in 1.0 min).
- the secondary bacteria culture was completed.
- the seed liquid culture time is generally 16-24 hours.
- the culture temperature is controlled at 29.0 ⁇ 1.0 °C
- the tank pressure is controlled at 0.050 ⁇ 0.010MPa
- the DO is controlled by manually adjusting the ventilation, oxygen and speed.
- the pH is controlled at 5.0.
- the initial flow rate of the glycerin solution was 1.7 mL / min (1s / 60s).
- the acceleration of the glycerin feed flow was adjusted according to the specific fermentation conditions, and the foam was manually replenished due to the hostile foam.
- the wet bacteria weight of the fermentation broth was sampled.
- the glycerol feeding was stopped and starvation was started.
- the carbon source was insufficient at this time, and the dissolved oxygen increased again. Adjust the ventilation volume and decrease the stirring speed to control the DO at 30% -40%.
- the starvation phase begins, and the starvation state is controlled for 1.0h.
- the automatic feeding system was started. The initial flow rate was 1.7 mL / min (1s / 60s), and the flow rate increased to 3.4 mL / min (2s / 60s) after 3 hours, and the flow rate increased to 5.1 mL / min after 6 hours. (3s / 60s), after 9h, it will enter the methanol induction stage, and increase the methanol flow acceleration according to the actual situation of DO.
- the acceleration of methanol flow is generally controlled within 13.6mL / min (8s / 60s), and the methanol induction time is generally controlled within 40-48h.
- DO should be controlled to 20-35%, and it is determined that there is no cumulative excess of methanol during this phase.
- the induction time is 40-48h or the volume of methanol replenishment is 20-30L, the methanol induction phase ends and the tank is started.
- the fermentation broth was centrifuged at a speed of 2000 rpm and centrifuged for 90 minutes for solid-liquid separation. The supernatant was collected, and the final solution was added with a solid concentration of 0.8M-1.0M sodium chloride. After it was completely dissolved, it was hollowed through 0.2 ⁇ m.
- a fiber membrane filtration system removes residual bacteria, cell debris, and especially the green pigment in Pichia fermentation broth; collects 0.2 ⁇ m filtrate to refine purification by phenyl hydrophobic chromatography.
- Phase A 1M sodium chloride solution
- B purified water plus 10% isopropanol, 0.5M sodium hydroxide solution to adjust the pH to 8.0.
- Phase A is equilibrated with a Phenyl Sepharose 6FF (GE company) high flow rate phenyl hydrophobic agarose gel chromatography column.
- the conductivity is 80 mS / cm after equilibrium is completed, the collected 0.2 ⁇ m filtrate is applied to the column. Wash about 3 column volumes with phase A flow, wait for UV absorption to drop below 500mV, elute with phase B, and collect the eluate.
- CM cation exchange chromatography purification Prepare mobile phase A: 20mM phosphate buffer, pH 7.6-7.8, conductivity 2.0-2.3mS / cm; mobile phase B: 20mM phosphate buffer, 1M NaCl , Between pH7.6-7.8.
- the recombinant human lysozyme sample in 3.2.2. was applied to a CM Sepharose and FF (GE company) high flow rate cation exchange agarose gel chromatography column.
- phase A was equilibrated to 2-3 column volumes, and the ladder After gradient 16% phase B elutes and removes impurities, it is adjusted to gradient 25% phase B elution, and the eluate is collected as recombinant human lysozyme.
- the eluate was purified by chromatography using a Sephadex G25 (GE company) gel, and the protein peak solution was collected and freeze-dried to obtain a recombinant human lysozyme lyophilized powder with a purity of more than 98%.
- the recombinant human lysozyme prepared in Example 1 is a white or off-white amorphous powder; it is odorless; it is easily destroyed in the presence of alkali, easily soluble in an aqueous solution of sodium chloride above 0.6%, and insoluble in acetone or ether.
- MALDI-TOF detected the molecular weight of recombinant human lysozyme 14700D ⁇ 100D.
- the recombinant human lysozyme protein prepared in Example 1 has a molecular weight of 14700D.
- the 15 amino acid sequence of the N-terminus of the recombinant human lysozyme is:
- the methanol content is not higher than 0.002%.
- the protein per 100 ⁇ g should not be higher than 10 ng.
- yeast protein bacterial cell protein residue determination method (“People's Republic of China 2015”
- General Principle 3414 it should not be higher than 0.1% of the total protein.
- Example 1 0.2 g of the recombinant human lysozyme prepared in Example 1 was measured and determined by the cautery residue inspection method ("People's Republic of China 2015” Third Edition General Principle 0841), and the residual residue did not exceed 4.0%.
- Reagent preparation phosphate buffer solution (0.02M / L pH6.5): Weigh 3.115g of Na 2 HPO 4 ⁇ 12H 2 O, weigh 1.763g of NaH 2 PO 4 ⁇ 2H 2 O, weigh NaCl 6.0 g, add 800mL of distilled water to dissolve, and make up to 1000mL.
- Substrate suspension Weigh 20mg of M. lysoderma powder, add 0.5mL of phosphate buffer, soak for 1 hour, and then add an appropriate amount of phosphate buffer to make the suspension measured at a wavelength of 450nm at 25 ° C The absorbance is 0.800 ⁇ 0.05 (prepared just before use).
- Recombinant human lysozyme solution Precisely weigh 10mg of recombinant human lysozyme, place it in a 5mL volumetric flask, add phosphate buffer to volume to 5mL, and then dilute to 200 ⁇ g / mL of recombinant human lysozyme solution.
- Pipette 0.1mL of a recombinant human lysozyme solution at a concentration of 200 ⁇ g / mL add it to a cuvette containing 3mL of substrate suspension at room temperature 25 °C, pH 6.5, place it in a UV spectrophotometer, and measure at a wavelength of 450nm Absorption, and calculate its potency. Each minute causes a decrease in absorbance of 0.001 as a unit of enzyme activity.
- W is the weight of the test article in the measurement solution, and the unit is ⁇ g.
- Total bacterial count (CFU / g): Take 0.5g and determine it according to the microbial limit inspection method of non-sterile products ("People's Republic of China" 2015 edition three general rules 1105). Total bacteria (CFU / g) ⁇ 100.
- CFU / g Total number of molds and yeasts
- the lyophilized powder of recombinant human lysozyme was less than 10 EU per 1 mg.
- the recombinant human lysozyme prepared in Example 1 meets various standards, is a qualified product, and can be further used to prepare artificial tears.
- Example 3 Study on Solubility of Recombinant Human Lysozyme and Sodium Chloride Content in Solution
- the test and statistical results of the recombinant human lysozyme solubility and the sodium chloride content in the solution are shown in Table 2. From the test data, it can be seen that in the solution below 0.6% sodium chloride content (0.4%, 0.2%, water for injection), the solution is an unclear and transparent liquid, and the overall state is a suspension. As the salt concentration decreases, the reorganization The suspension state of human lysozyme gradually increased, and the suspension state of recombinant human lysozyme in pure water was the most obvious. At the same time, the protein content and bacteriostatic activity also showed a decreasing trend, mainly because the recombinant human lysozyme was less than 0.6% chlorinated. The sodium content solution is not completely dissolved.
- Example 4 Relationship between bacteriostatic activity of recombinant human lysozyme and pH
- Phosphate buffer solutions corresponding to pH were diluted into recombinant human lysozyme solutions with a content of 1.5 mg / mL and 0.75 mg / mL, respectively, thereby obtaining 3 kinds of contents, each content corresponding to 7 recombinant human lysozyme solutions with different pH,
- the biological activity-titer assay method in Example 2 was used (the phosphate buffer solution in the assay was weighed to prepare the corresponding disodium hydrogen phosphate and sodium dihydrogen phosphate at different pHs in this test to prepare). Antibacterial activity.
- the bacteriostatic activity of the recombinant lysozyme decreased with the change of pH, and the closer the pH was, the closer The isoelectric point of recombinant human lysozyme was 9.24. The lower the solubility and stability, the more obvious the antibacterial activity decreased.
- the recombinant human lysozyme is the recombinant human lysozyme prepared in Example 1;
- Sodium hyaluronate was purchased from Huaxi Furuida Bio-Pharmaceutical Co., Ltd., an artificial tear-grade sodium hyaluronate bulk drug, and its national standard H20113379.
- Disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, and sodium chloride were purchased from Sinopharm Group.
- composition of the artificial tear fluid in this example is shown in Table 4.
- the artificial tear fluid is filtered and sterilized with a 0.22 ⁇ m sterilizing filter under the condition of a filtering operating pressure of 0.35-0.40 MPa, and then the sterile medicinal solution is transferred to a sterilized material storage tank.
- the sterilized medicinal solution is poured into a container and sealed under a level A laminar flow protection by a blow-fill-seal integrated machine, and the filling specification is 0.8 mL / branch. Subsequently, the aseptically filled products are inspected, packaged, and stored.
- the artificial tear solution of this embodiment is packed in a daily dose, which is used immediately when used, and it is used up within 24 hours.
- composition of artificial tears of Examples 6 to 9 is shown in Table 4.
- Example 5 Example 6
- Example 7 Example 8
- Example 9 Recombinant human lysozyme 15.0 60.0 7.5 15.0 60.0 Sodium hyaluronate 10.0 60.0 10.0 30.0 20.0 Disodium hydrogen phosphate dodecahydrate 20.0 40.0 20.0 20.0 40.0 Sodium dihydrogen phosphate dihydrate 9.0 18.0 9.0 9.0 18.0 Sodium chloride 75.0 140.0 76.0 72.0 148.0 Water for Injection 9871.0 19682.0 9877.5 9854.0 19714.0
- the artificial tears of Examples 5 to 9 are all colorless and clear liquids.
- the labeled volume is 0.8mL / piece, the average volume is not less than the labeled volume, and each container volume is not less than the labeled volume.
- Reagent preparation phosphate buffer solution (0.02M / L pH6.5): Weigh 3.115g of Na 2 HPO 4 ⁇ 12H 2 O, weigh 1.763g of NaH 2 PO 4 ⁇ 2H 2 O, weigh NaCl 6.0 g, add 800mL of distilled water to dissolve, and make up to 1000mL.
- Substrate suspension Weigh 20mg of M. lysoderma powder, add 0.5mL of phosphate buffer, soak for 1 hour, and then add an appropriate amount of phosphate buffer to make the suspension measured at a wavelength of 450nm at 25 ° C The absorbance is 0.800 ⁇ 0.05 (prepared just before use).
- Recombinant human lysozyme solution Take samples of each example, place them in 5mL volumetric flasks, add phosphate buffer to volume to 5mL, and dilute to 200 ⁇ g / mL recombinant human lysozyme solution.
- W is the weight of the test article in the measurement solution, and the unit is ⁇ g.
- the biological activity test was 70% -200% of the indicated amount.
- test solution Take 1-2 artificial tears, accurately measure 0.7mL, place in a 10mL volumetric flask, dilute to the mark with water for injection, and shake well.
- UV-visible spectrophotometry method According to the ultraviolet-visible spectrophotometry method (General Principles 0401 of the Third Edition of the Pharmacopoeia of the People's Republic of China 2015), 0 tube is blank, the absorbance is measured at a wavelength of 530nm, and the corresponding absorbance is calculated by the ⁇ g number of glucuronic acid. .
- the measurement was repeated three times, and the average value was calculated to calculate the sodium hyaluronate content.
- the content of sodium hyaluronate should be 90% -110% of the indicated amount.
- Test bacteria Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli.
- Blank control a preparation prepared according to Example 5 without adding recombinant human lysozyme
- Test article artificial tears, prepared according to Example 5, with an activity of 2.19 ⁇ 10 5 U / mL;
- Bacterial preparation LB medium, weigh 0.5g yeast extract, 1.0g peptone, 1.0g sodium chloride, dissolve in 100mL deionized water, sterilize at 121 °C for 30 minutes, inoculate test bacteria into LB culture Incubate at 37 ° C and 200 rpm overnight.
- the bacteriostatic effect test of the LB agarose culture plate paper sheet diffusion bacteriostatic method is shown in Fig. 1a to Fig. 1d, and the statistical results are shown in Table 5. It can be seen from the size of the bacteriostatic zone that the control, blank control, and test product have no obvious antibacterial effect on gram-negative bacteria Escherichia coli; compared to 0.1% sodium hyaluronate eye drops and blank control And artificial tears have obvious antibacterial effects on Gram-positive bacteria Staphylococcus aureus, Micrococcus luteus, and Bacillus subtilis.
- Example 12 Long-term stability and accelerated test of artificial tears
- Artificial tears are tested for stability under the storage conditions specified in the market.
- the stability characteristics of artificial tears during transportation and storage are investigated to serve as a basis for determining the validity period and storage conditions.
- the artificial tear fluid sample prepared in Example 5 had an activity of 2.19 ⁇ 10 5 U / mL, and was stored at 4 ° C., 25 ° C., 37 ° C., and a humidity of 45% to 65%, protected from light. Sampling is performed regularly for traits, pH, osmotic pressure, sterility, and activity testing. Long-term stability test samples are tested monthly for the first 6 months in the first year, every 3 months for the next 6 months, and every 6 months in the second year. 37 ° C accelerated test samples are tested monthly. The statistical results of the stability tests at 4 ° C, 25 ° C, and 37 ° C are shown in Table 6, Table 7, and Table 8, respectively.
- Example 13 Pharmacodynamic test of artificial tears in rabbit eyes with excessive evaporation of dry eye model with corneal epithelial cell damage
- test and reference materials provided are used directly and do not require preparation.
- Rabbit eyes are convenient for slit lamp microscopy and are commonly used animal models to study dry keratoconjunctivitis (KCS).
- KCS dry keratoconjunctivitis
- the common method of establishing rabbit KCS is to surgically remove the lacrimal gland, Hastelloy gland and the third eyelid, and burn the conjunctiva of New Zealand rabbits with 30% trichloroacetic acid.
- Sex and quantity 18 females and 18 males.
- the room temperature is controlled at 20-25 ° C, and the humidity is 40-70%. 12h lighting, 12h darkness. New Zealand rabbits are housed in stainless steel cages, one per cage, and the cages are cleaned once a day.
- Random numbers were used to group the animals into groups, and redundant animals were eliminated. Each group was assigned a single experimental animal number. The test animal number is used for identification in the original data. The cage card is marked with the animal number, dose group and sex.
- the lacrimal gland, Hastellus gland and the third eyelid were surgically removed, and the conjunctiva of New Zealand rabbits was burned with 30% trichloroacetic acid to establish a model.
- the left eye of each rabbit was operated to establish a model, and the right eye was used as a self-control.
- Each operation of the TFT inspection is performed by the same person, and the tear fluid and smear operations are performed by a double-blind method.
- the examination time is from 10:00 to 12:00 every day.
- the graphic grading method of Rolando et al. According to the integrity, uniformity, and branching state of tear-fermented crystals, it is divided into 4 levels.
- Corneal epifluorescein staining (corneal, fluorescent, staining, FL)
- a 100 g / L sodium fluorescein solution was dripped into the conjunctival sac, and examined under a cobalt lamp under a slit lamp to observe the corneal epithelial infection.
- the scoring method refers to the consensus of experts on dry eye clinical diagnosis and treatment (2013).
- the 12-point method is adopted: the cornea is divided into 4 quadrants, each quadrant is 0-3 points, no staining is 0 points, and 1-30 point-like staining is 1 Scores,> 30 spot-like staining but no staining was fused to 2 points, and 3 points were corneal spot-like coloring fusion, filamentous, and ulcers.
- mice were randomly grouped.
- the test consists of 6 groups, namely the blank control group, the recombinant human lysozyme reference group, the artificial tear low-dose group (0.75 mg / mL), the middle-dose group (1.50 mg / mL), and the high-dose group (3.00 mg / mL).
- sodium hyaluronate eye drops in the control group 3 males and 36 females in each group.
- Frequency Dosed daily at 8 o'clock, 12 o'clock, and 16 o'clock, 3 times a day.
- Volume 2 drops (about 70 ⁇ L) / eye.
- tear secretion test In 4 weeks of continuous administration, tear secretion test, tear fern-like crystal test, and sodium fluorescein staining under a slit lamp were performed before the administration, one week, two weeks, three weeks, and four weeks (methods as above). At the end of the test, the cornea and conjunctiva were taken for HE staining, and histological observation was performed under a microscope.
- the measurement data were calculated as the average and standard deviation of each group, and then the analysis of variance was performed.
- ac in FIG. 2 is a sequence before operation, 1 week after operation, and 2 weeks after operation.
- ac in FIG. 3 is a sequence before operation, 1 week after operation, and 2 weeks after operation.
- the tear secretion test the tear fern-like crystal test, the fluorescein staining observation, the difference between the model group and the control group was very significant, all of which were statistically significant, indicating that the modeling was successful.
- the fern-like crystals of the high-concentration group, the medium-concentration group, the low-concentration group, and the sodium hyaluronate eye control group gradually increased after 1-4 weeks of treatment, and there was a significant difference compared with the blank control group (p ⁇ 0.05), which was statistically significant.
- the corneal epithelium of New Zealand rabbits was observed under an optical microscope after HE staining.
- the corneal epithelium in the blank group became thinner and the cells were arranged disorderly; the low-concentration group, the recombinant human lysozyme control group and the sodium hyaluronate eye drops control
- the corneal epithelium of the group was thinner, and the number of cells was small, and there was no significant difference.
- the corneal epithelium thickness and cell number of the high-concentration and medium-concentration groups recovered well, compared with the sodium hyaluronate eye drops control group and the recombinant human lysozyme control group.
- Significant difference (results are shown in Fig. 12, af in Fig. 12 are high concentration group, medium concentration group, low concentration group, recombinant human lysozyme reference group, blank control group, sodium hyaluronate control group) .
- artificial tears can significantly improve the amount of tear secretion, tear secretion properties, reduce the degree of corneal damage, and protect the corneal epithelium in a New Zealand rabbit model of dry eye secretion-type dry eye syndrome.
- Sodium hyaluronate eye drops control group and recombinant human lysozyme reference group artificial tears, especially the high-concentration group (3.00mg / mL), medium-concentration group (1.50mg / mL) can more effectively improve the lack of tear secretion
- the amount of tear secretion and the nature of tear secretion in a New Zealand rabbit model of dry eye syndrome reduce the corneal damage and promote the repair of corneal damaged cells.
- Table 11 Corneal epithelial fluorescein sodium staining scores in surgical eyes and control eyes
- n 6. Compared with the blank control group, * p ⁇ 0.05, ** p ⁇ 0.01; compared with the sodium hyaluronate eye drops control group, # p ⁇ 0.05; compared with the recombinant human lysozyme control group, ⁇ p ⁇ 0.05.
- n 6. Compared with the blank control group, * p ⁇ 0.05, ** p ⁇ 0.01; compared with the sodium hyaluronate eye drops control group, # p ⁇ 0.05; compared with the recombinant human lysozyme control group, ⁇ p ⁇ 0.05.
- n 6. Compared with the blank control group, * p ⁇ 0.05, ** p ⁇ 0.01; compared with the sodium hyaluronate eye drops control group, # p ⁇ 0.05; compared with the recombinant human lysozyme control group, ⁇ p ⁇ 0.05.
- Example 14 Human dry eye pharmacodynamics test with artificial tears
- Test article artificial tears
- test personnel were selected, including 28 males and 36 females, aged 23-50 years.
- the selection criteria were: 1Careers who have faced computer work for a long time and have excessive eyes, such as finance, legal affairs, office administration, quality Light inspectors at inspection posts; young employees who have used computers and mobile phones for a long time; 2 obvious eye redness, itching, dry eyes, photophobia, eye fatigue, foreign body sensation, burning sensation, soreness, People with conscious discomfort such as eye pain.
- 3Excluded objects People with systemic diseases who have used glucoseboids, non-steroidal anti-inflammatory drugs and immunosuppressants.
- test subjects performed a dry eye symptom questionnaire before and after treatment.
- the questionnaire score items included redness, itching, dry eyes, photophobia, eye fatigue, foreign body sensation, burning sensation, soreness, and eye pain.
- the left and right eyes were calculated by their severity and duration from light to severe with 0-4 points. All data were analyzed statistically using SPSS20 software. The data were expressed as mean ⁇ standard deviation and compared using t test. P ⁇ 0.05 is considered statistically significant.
- the artificial tear solution for the test can significantly and effectively improve the symptoms associated with dry eye syndrome caused by insufficient tear secretion due to excessive eye use in people's work and life, and reduced number of blinks, and promote corneal damage Cell repair.
- n 64. Compared with left eye, * p ⁇ 0.05.
Abstract
Description
实施例5 | 实施例6 | 实施例7 | 实施例8 | 实施例9 | |
重组人溶菌酶 | 15.0 | 60.0 | 7.5 | 15.0 | 60.0 |
透明质酸钠 | 10.0 | 60.0 | 10.0 | 30.0 | 20.0 |
十二水合磷酸氢二钠 | 20.0 | 40.0 | 20.0 | 20.0 | 40.0 |
二水合磷酸二氢钠 | 9.0 | 18.0 | 9.0 | 9.0 | 18.0 |
氯化钠 | 75.0 | 140.0 | 76.0 | 72.0 | 148.0 |
注射用水 | 9871.0 | 19682.0 | 9877.5 | 9854.0 | 19714.0 |
时间 | 温度 | 性状 | pH | 渗透压(mOsmol/kg) | 无菌 | 活性(U/mL) |
0月 | - | 无色澄清液体 | 6.50 | 299.2 | 无菌 | 2.19×10 5 |
1月 | 4℃ | 无色澄清液体 | 6.52 | 300.0 | 无菌 | 2.16×10 5 |
2月 | 4℃ | 无色澄清液体 | 6.52 | 299.2 | 无菌 | 2.18×10 5 |
3月 | 4℃ | 无色澄清液体 | 6.48 | 298.4 | 无菌 | 2.20×10 5 |
4月 | 4℃ | 无色澄清液体 | 6.50 | 300.0 | 无菌 | 2.16×10 5 |
5月 | 4℃ | 无色澄清液体 | 6.48 | 299.2 | 无菌 | 2.19×10 5 |
6月 | 4℃ | 无色澄清液体 | 6.48 | 298.4 | 无菌 | 2.21×10 5 |
9月 | 4℃ | 无色澄清液体 | 6.50 | 300.0 | 无菌 | 2.17×10 5 |
12月 | 4℃ | 无色澄清液体 | 6.52 | 300.0 | 无菌 | 2.17×10 5 |
18月 | 4℃ | 无色澄清液体 | 6.48 | 300.0 | 无菌 | 2.21×10 5 |
24月 | 4℃ | 无色澄清液体 | 6.50 | 298.4 | 无菌 | 2.18×10 5 |
时间 | 温度 | 性状 | pH | 渗透压(mOsmol/kg) | 无菌 | 活性(U/mL) |
0月 | - | 无色澄清液体 | 6.50 | 299.2 | 无菌 | 2.19×10 5 |
1月 | 25℃ | 无色澄清液体 | 6.52 | 301.6 | 无菌 | 2.18×10 5 |
2月 | 25℃ | 无色澄清液体 | 6.48 | 300.0 | 无菌 | 2.18×10 5 |
3月 | 25℃ | 无色澄清液体 | 6.48 | 299.2 | 无菌 | 2.19×10 5 |
4月 | 25℃ | 无色澄清液体 | 6.52 | 300.0 | 无菌 | 2.20×10 5 |
5月 | 25℃ | 无色澄清液体 | 6.48 | 300.0 | 无菌 | 2.19×10 5 |
6月 | 25℃ | 无色澄清液体 | 6.48 | 299.2 | 无菌 | 2.18×10 5 |
9月 | 25℃ | 无色澄清液体 | 6.50 | 300.0 | 无菌 | 2.19×10 5 |
12月 | 25℃ | 无色澄清液体 | 6.52 | 300.0 | 无菌 | 2.20×10 5 |
18月 | 25℃ | 无色澄清液体 | 6.52 | 299.2 | 无菌 | 2.17×10 5 |
24月 | 25℃ | 无色澄清液体 | 6.52 | 300.0 | 无菌 | 2.18×10 5 |
时间 | 温度 | 性状 | pH | 渗透压(mOsmol/kg) | 无菌 | 活性(U/mL) |
0月 | - | 无色澄清液体 | 6.50 | 299.2 | 无菌 | 2.19×10 5 |
1月 | 37℃ | 无色澄清液体 | 6.48 | 300.0 | 无菌 | 2.20×10 5 |
2月 | 37℃ | 无色澄清液体 | 6.48 | 299.2 | 无菌 | 2.18×10 5 |
3月 | 37℃ | 无色澄清液体 | 6.50 | 298.4 | 无菌 | 2.19×10 5 |
4月 | 37℃ | 无色澄清液体 | 6.52 | 300.0 | 无菌 | 2.17×10 5 |
5月 | 37℃ | 无色澄清液体 | 6.48 | 299.2 | 无菌 | 2.18×10 5 |
6月 | 37℃ | 无色澄清液体 | 6.52 | 298.4 | 无菌 | 2.17×10 5 |
7月 | 37℃ | 无色澄清液体 | 6.50 | 301.6 | 无菌 | 2.16×10 5 |
8月 | 37℃ | 无色澄清液体 | 6.48 | 305.0 | 无菌 | 2.08×10 5 |
9月 | 37℃ | 无色澄清液体 | 6.48 | 308.0 | 无菌 | 1.78×10 5 |
10月 | 37℃ | 无色澄清液体 | 6.48 | 310.2 | 无菌 | 1.56×10 5 |
11月 | 37℃ | 无色澄清液体 | 6.46 | 322.6 | 无菌 | 1.03×10 5 |
12月 | 37℃ | 无色澄清液体 | 6.46 | 345.6 | 无菌 | 1.01×10 5 |
组别 | n | 术前 | 术后1周 | 术后2周 |
手术眼 | 36 | 19.67±3.70 | 9.94±2.37** | 3.44±1.05** |
对照眼 | 36 | 19.41±3.05 | 19.97±3.00 | 18.46±2.22 |
组别 | n | 术前 | 术后1周 | 术后2周 |
手术眼 | 36 | 1.08±0.28 | 2.53±0.91** | 3.53±0.65** |
对照眼 | 36 | 1.11±0.32 | 1.08±0.28 | 1.14±0.35 |
组别 | n | 术前 | 术后1周 | 术后2周 |
手术眼 | 36 | 2.39±1.02 | 6.92±2.49** | 9.52±1.56** |
对照眼 | 36 | 2.52±1.75 | 3.03±1.25 | 3.39±1.24 |
治疗前 | 治疗3天 | 治疗7天 | 治疗14天 | |
左眼 | 18.3±3.46 | 18.6±2.87 | 16.6±3.55 | 13.6±2.76 |
右眼 | 19.5±2.16 | 15.6±3.75 | 11.7±3.25 * | 9.68±4.14 * |
Claims (10)
- 一种含有重组人溶菌酶的新型人工泪液,其包括主成分和辅料,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%。
- 根据权利要求1所述的新型人工泪液,其中,所述重组人溶菌酶与人体天然溶菌酶的氨基酸序列是完全一致的。
- 根据权利要求1所述的新型人工泪液,其中,以新型人工泪液的总质量计,所述重组人溶菌酶的含量为0.150%-0.300%。
- 根据权利要求1所述的新型人工泪液,其中,所述新型人工泪液的pH值为6.4-6.6,优选为6.5。
- 根据权利要求1所述的新型人工泪液,其中,所述辅料包括pH稳定剂;优选地,所述pH稳定剂包括柠檬酸钠、碳酸钠、碳酸氢钠、枸橼酸钠或者由磷酸氢二钠与磷酸二氢钠构成的缓冲液;更优选地,所述pH稳定剂为由磷酸氢二钠与磷酸二氢钠构成的缓冲液;优选地,所述磷酸氢二钠为十二水合磷酸氢二钠,所述磷酸二氢钠为二水合磷酸二氢钠,更优选地,二者的质量比为20:9。
- 根据权利要求1所述的新型人工泪液,其中,所述新型人工泪液的渗透压摩尔浓度为285-310mOsmol/kg。
- 根据权利要求1、5或6所述的新型人工泪液,其中,所述辅料包括氯化钠;优选地,以所述新型人工泪液的总质量计,所述氯化钠的含量为0.70%-0.76%,氯化钠的含量以纯固体计。
- 根据权利要求1-7任一项所述的新型人工泪液,其中,按照所述新型人工泪液的总量为10000-20000重量份计,所述新型人工泪液包括:重组人溶菌酶7.5-60.0份,透明质酸钠10.0-60.0份,十二水合磷酸氢二钠20.0-40.0份,二水合磷酸二氢钠9.0-18.0份,氯化钠70.0-152.0份,注射用水9841.0-19755.0份。
- 根据权利要求8所述的新型人工泪液,其中,按照所述新型人工泪液的总量为10000重量份计,所述新型人工泪液包括:重组人溶菌酶15.0-30.0份,透明质酸钠10.0-30.0份,十二水合磷酸氢二钠20.0份,二水合磷酸二氢钠9.0份,氯化钠70.0-75.0份,注射用水9841.0-9871.0份。
- 根据权利要求8或9所述的新型人工泪液,其中,按照所述新型人工泪液的总量为10000重量份计,所述新型人工泪液包括:重组人溶菌酶7.5份,透明质酸钠10.0份,十二水合磷酸 氢二钠20.0份,二水合磷酸二氢钠9.0份,氯化钠76.0份,注射用水9877.5份;或者,按照所述新型人工泪液的总量为10000重量份计,所述新型人工泪液包括:重组人溶菌酶15.0份,透明质酸钠10.0份,十二水合磷酸氢二钠20.0份,二水合磷酸二氢钠9.0份,氯化钠75.0份,注射用水9871.0份;或者,按照所述新型人工泪液的总量为10000重量份计,所述新型人工泪液包括:重组人溶菌酶15.0份,透明质酸钠30.0份,十二水合磷酸氢二钠20.0份,二水合磷酸二氢钠9.0份,氯化钠72.0份,注射用水9854.0份;或者,按照所述新型人工泪液的总量为20000重量份计,所述新型人工泪液包括:重组人溶菌酶60.0份,透明质酸钠20.0份,十二水合磷酸氢二钠40.0份,二水合磷酸二氢钠18.0份,氯化钠148.0份,注射用水19714.0份;或者,按照所述新型人工泪液的总量为20000重量份计,所述新型人工泪液包括:重组人溶菌酶60.0份,透明质酸钠60.0份,十二水合磷酸氢二钠40.0份,二水合磷酸二氢钠18.0份,氯化钠140.0份,注射用水19682.0份。
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US16/948,830 US20210121540A1 (en) | 2018-06-14 | 2020-10-01 | Novel artificial tears containing recombinant human lysozyme |
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CN201810614434.9A CN110604812B (zh) | 2018-06-14 | 2018-06-14 | 一种含有重组人溶菌酶的人工泪液 |
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US (1) | US20210121540A1 (zh) |
EP (1) | EP3834839B1 (zh) |
CN (1) | CN110604812B (zh) |
AU (2) | AU2018428270B2 (zh) |
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CN102552889A (zh) * | 2010-12-27 | 2012-07-11 | 上海高科联合生物技术研发有限公司 | 一种生物杀菌滴眼液制剂 |
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US6569903B2 (en) * | 1999-12-07 | 2003-05-27 | Rohto Pharmaceutical Co., Ltd. | Ophthalmic compositions |
US6991824B2 (en) * | 2000-05-02 | 2006-01-31 | Ventria Bioscience | Expression of human milk proteins in transgenic plants |
CN1193768C (zh) * | 2002-10-01 | 2005-03-23 | 青岛海洋大学 | 一种人工泪液 |
JP4453815B2 (ja) * | 2003-12-08 | 2010-04-21 | ライオン株式会社 | ドライアイ治療剤 |
GB0404693D0 (en) * | 2004-03-02 | 2004-04-07 | Univ London | Pharmaceutical preparations for the treatment of ocular surface and other disorders |
JP5117384B2 (ja) * | 2005-07-01 | 2013-01-16 | シグマ−タウ・インドゥストリエ・ファルマチェウチケ・リウニテ・ソシエタ・ペル・アチオニ | 点眼剤の形態における眼用生理学的サプリメントまたは医薬の調製のためのl−カルニチンまたはアルカノイルl−カルニチンの使用 |
AR062046A1 (es) * | 2006-07-25 | 2008-08-10 | Osmotica Pharmaceutical Argentina S A | Soluciones oftalmicas |
US20080213188A1 (en) * | 2007-03-02 | 2008-09-04 | Saint Simeon Lda | Novel ophthalmic compositions containing human recombinant lysozyme and use thereof for treating eye conditions and as contact lens solutions |
US8673937B2 (en) * | 2008-04-23 | 2014-03-18 | Otsuka Pharmaceutical Co., Ltd. | Eye-drop preparation and use thereof |
CN102188695B (zh) * | 2010-03-03 | 2014-01-22 | 上海昊海生物科技股份有限公司 | 一种眼用凝胶组合物 |
CN103182074B (zh) * | 2011-12-30 | 2016-03-30 | 沈阳兴齐眼药股份有限公司 | 一种含有溶菌酶的眼用制剂 |
CN106265720A (zh) * | 2015-05-12 | 2017-01-04 | 上海昊海生物科技股份有限公司 | 一种复合人工泪液及其制备方法 |
CN107913202A (zh) * | 2017-09-29 | 2018-04-17 | 上海雨淑缇化妆品有限公司 | 一种眼球明眸调理营养液及其制备方法 |
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CN102552889A (zh) * | 2010-12-27 | 2012-07-11 | 上海高科联合生物技术研发有限公司 | 一种生物杀菌滴眼液制剂 |
Non-Patent Citations (2)
Title |
---|
"Pharmacopoeia of People's Republic of China", vol. III, 2015, article "the first method: trypsin lysis-reversed phase high performance liquid chromatography" |
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US20210121540A1 (en) | 2021-04-29 |
AU2018428270B2 (en) | 2022-09-01 |
EP3834839B1 (en) | 2023-11-22 |
AU2018428270A1 (en) | 2020-10-22 |
EP3834839C0 (en) | 2023-11-22 |
EP3834839A1 (en) | 2021-06-16 |
CN110604812A (zh) | 2019-12-24 |
EP3834839A4 (en) | 2022-03-30 |
HK1252301A2 (zh) | 2019-05-24 |
AU2019100500A4 (en) | 2019-06-06 |
CN110604812B (zh) | 2023-01-31 |
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