Study On The Preparation Of Lysozyme Eye Drops And Its Effect On Rabbit Model Of Bacterial Keratitis

The common infectious eye diseases in clinic,such as conjunctivitis and keratitis,are mainly caused by bacterial infection,and bacterial keratitis is the first reason causing corneal blindness in developing countries.A large number of clinical etiology analysises showed that the Gram-positive coccobacteria such as Staphylococcus epidermidis,Staphylococcus aureus, Micrococcus,etc.are the main pathogens,followed by the Gram-negative bacteria such as Pseudomonas aeruginosa,E.coli etc.The proportion of the former has the trend of increasing year by year and that of the latter has the trend of declining.The most commonly used medicines in the treatment of bacterial infectious eye diseases were antibiotic eye drops, which were easy to develop drug resistance.With the enlarging use of antibiotics,the number of drug-resistant strains was increasing.So antibiotic eye drops should not be used in long-term because of its side effects.Therefore,the study on non-antibiotic eye drops for bacterial infectious eye diseases is a research hotspot.Lysozyme,an enzyme existing in human normal body fluids and tissues as a non-specific immune factor,has a variety of pharmacological effects,such as antibacterial,antiviral and anti-tumor effects.It can effectivly hydrolyze the peptidoglycan of bacterial cell wall.The reaction is the hydrolysis of theβ-1,4 glycosidic bond between N-acetylglucosamine(NAG) and N-acetylmuramic acid(NAM).Lysozyme displays high decomposition effects on Gram-positive bacteria,such as aerobic spore-forming bacteria,Bacillus subtilis and Micrococcus radiodurans.Lysozyme also has a certain decomposition effects on E.coli, Variable cocci,Vibrio parahaemolyticus and other Gram-negative bacteria.As a alkaline protein,lysozyme with high positive charge can bind to viral proteins with negative charge in vivo,and it can form compound salt with DNA,RNA and apolipoprotein,which lead to the inactivation of invasive viruses.Lysozyme has many advanteges such as,good biocompatibility,low drug resistance,no tissue stimulation and non-toxicity and so on.Egg white lysozyme,a typical representative of lysozyme,has very stable chemical properties, such as it is stable to heat and its structure is almost unchanged in the pH range of 1.2～11.3. Therefore,the main indicator of the study was the lysozyme activity.The possible adjuvants for lysozyme eye drops were screened and the possible formula was optimized.After that,the effective lysozyme concentration and the stability of theeye drops were studied by testing the antibacterial activity.The effect of lysozyme was verified in rabbit keratitis model.All the results were summarized as follows.1 Characterization of egg white lysozymeThe Mr of egg white lysozyme was approximately 14 kD.The lysozyme had the highest enzyme activity during the pH of 6～6.5,while a relatively stable activity was obtained in the pH range of 5～7,but a sharp activity decrease appeared when the pH＞7.Lysozyme was stable in the temperature range of 25～65℃,but the activity decreasd when the temperature was higher than 65℃,especially when the temperature was more than 75℃.2 Compatibility study on egg white lysozyme,ingredients and adjuvants commonly used in ophthalmic preparationsThe ingredients and adjuvants with inhibition activiy on lysozyme activity were Ca²⁺, Co²⁺,Fe²⁺,Zn²⁺,Cu²⁺,Tween-20,Tween -80,glycerol,EDTA-2Na with a concentration of higer than 0.3%,ethanol with a concentration of higer than 5%,aminoethylsulfonic acid with a concentration of higer than 0.25%,vitamin B6 with a concentration of higer than 0.5%and borax with a concentration of higer than 0.05%.The ingredients and adjuvants with no effects on lysozyme activity were propylene glycol,borneol and aspartic acid.The ingredients and adjuvants with enchancing activity on lysozyme activity were Na⁺,Mn²⁺,K⁺,Mg²⁺,0.0005% - 0.3%EDTA-2Na,0.05%- 2%boric acid,0.012%- 0.04%menthol,0.5%-10%glycine, 0.012%～0.04%ethylparaben,0.05%-0.5%acetone chloroform and 0.03%benzalkonium bromide.3 Antibacterial activity study of egg white lysozymeLysozyme showed bactericidal effects on Micrococcus lysodeikticus at the concentration of 2.5μg/mL,on Bacillus cereus at 1.8 mg/mL,on Bacillus subtilis at 60μg/mL.With the presence of 0.01%EDTA-2Na,lysozyme could disinfect Staphylococcus aureus at the concentration of 3.3 mg/mL and Blastomyces albicans at 3 mg/mL.But,no obvious inhibition effects on Pseudomonas aeruginosa and E.coli were observed at the concentration of 7.5 mg/mL and 4mg/mL,respecitively.4 Formula study of lysozyme eye dropsAccording to the results of stability study,the final formula of lysozyme eye drops was composed of:lysozyme,5g;Na₂HPO₄,7.86g;NaH₂PO₄,10.4g;EDTA-2Na,0.5g;NaCl, 3.4g;menthol,0.25g;ethanol,5mL;benzalkonium bromide,0.2g;volume adjusted to 1000mL with water for injection.5 Establishment of quality standard of lysozyme eye dropsA quality standard for the lysozyme eye drops was established after research.6 Stability study on lysozyme eye dropsGood stability of the lysozyme eye drops was showen after preliminary study.7 Effects of lysozyme eye drops in rabbit bacterial keratitis modelLysozyme eye drops can effectively control Staphylococcus aureus- and Pseudomonas aeruginosa-induced corneal inflammation,and alleviate the symptoms.The number of bacteria decreased obviously after using the eye drops.8 Local irritation test of lysozyme eye dropsNo irritation on eyes was observed.In this study,results and conclusions which we have obtained are as follows:(1) The characterization and antibacterial activity of egg white lysozyme were studied and the formula screening was completed through the study on formula compatibility and stability.(2) Accoding to the standars established for lysozyme eye drops,good stability of lysozyme eye drops was got.(3) Lysozyme eye drops could effectively control Staphylococcus aureus- and Pseudomonas aeruginosa-induced corneal inflammation,alleviate the symptoms.The number of bacteria decreased obviously after using the eye drops.(4) No irritation of lysozyme eye drops on eyes was observed.