The Safety For Combination Usage Of Borneol With Some Preservatives In Ophthalmic Preparation

ObjectiveBorneol is a commonly drug in Ophthalmic preparation using in eye drops widely,which has an ef’f’ect of eliminating nebula to barrier.To ensure sepsis,preservative is often added into Ophthalmic preparation.Azone,which has an effect of penetration enhancing,for combining with preservative will strengthen ocular surface injury,rarely used in ophthalmic preparations.Borneol also has an effect of enhancing penetration,but its effect of combining with preservati ves is still not clear.This study was to investigate the in vitro cell injury and damage effect in rabbit of the combination of borneol and preservatives including Benzalkonium Chloride and Phenoxy ethanol,observing its distribution and excretion in conjunctival sac,evaluating the safety or the combination of borneol and preservatives in Ophthalmic preparation,study some part of its injury mechanism.Methods1.Draize eye irritation test or borneol and preservatives:Normal rabbits were dripped into borneol and commonly used preservatives,then observed the corneal and iris and evaluated the degree of eye irritation in 1、2、4、24 and 72 hours according to the score standard of draize irritation test.2.Hemolysis test:Got the blood from the rabbits car artery,prcepared into 2%Red Blood Cells(RBC)suspension,then divided into groups:borneol group,preservatives groups,combination of borneol and borneol groups and combination of borneol solvenL and preservatives groups,using normal salince as negative control and distilled water as positive control.Drawed the curve of drug’ s hemolysis concentration rate,analysed it with Isobolographic analysis and Design Expert to definite the type of the combination of borneol and preservatives.3.Rabbit corneal epithel ial cell experiment:When the rabbit corneal epithelial cell was cultured to logarithmic growth period,divided it into groups:borneol group,preservatives groups,combination of borneol and borneol groups and combination of borneol solvent and preservatives groups,taking culture medium as operation control.Observed the injury of the corneal epithelial cell and the trend of the recovery after administration.4.To establish detection methods to detect benzalkonium chl.oride(BAC)by liquid chromatography-mass spectrometry and phenoxy ethanol by gas chromatography-mass spectrometry in biological samples:the establishment of the detection,study on specificity,disposing biological samples(in cluding tears,cornea,sclera and iris).5.The metabolism of preservatives in conjunctival sac:50μL drugs(preservatives groups,combination of borneol and borneol groups and co--mbination of borneol solvent and preservatives groups)was dripped into Rabbits’ s eyes,then collected tear in 1、2.5、5、10、15、20、30、45、60 and 120 min.Detect the drugs’ concentration and draw the curve of pre servative in conjunctival sac.6.The ocular surface injury of borneol and preservatives:50μL drug s(normal saline group,borneol group,preservatives groups,combination of borneol and borneol groups and combination of borneol solvent and pre servatives groups)was dripped into Rabbits’ eyes twice a day.Tested t he tear secretion On the 0th,14th and 28th day,and tested the conjunctival impression cytology on 28th day.Rabbits were sacrificed on 30th day and eyes were removed and separated aqueous humor,cornea,sclera,iris,crys talline lens and Vitreous from eyes.Detected the drugs’ concentration and observe the injury by HE stain,PAS stain and electron microscopy.Results:1.Draize eye irritation test of borneol and preservatives:lmg/ml,borneol and commonly used concentration of preservatives’ scores were less than 3.9,meaning no injury to ocular surface.The preservatives was put in order from low to high according to the scores:0.05mg/mL Thimerosal,3mg/mL Phenoxy ethanol,0.1mg/mL Benzalkonium Chloride,0.3mg/mL Ethylparaben,5mg/mlL Chlorobutanol.2.Hemolysis test:At the clinical concentration of preservatives in ophthalmic preparation,the combination effect of borneol with ethylparaben and phenoxyethanol was rivalry,while the combination effect of borneol with chlorobutanol was addition,meaning that borneol would not add the hemolysis effect to these three drugs.Our previous experiments indicated that borneol combined with benzalkonium chloride’ s effect was Coordination.3.Rabbit corneal epithelial cel1 experiment:At the clinical concentr--ation of preservatives in Ophthalmic preparation,benzalkonium chloride and chlorobutano had serious injury on rabbit corneal epithelial cell,while phenoxyethanol and ethylparaben’ s irritation to rabbit cornenl cp-i the]ial cell was little.At the same time,the cytotoxicity of the combi--nation of borneol with phenoxyethanol and ethylparaben was little.The i--njured cell was continued to be cultured,after a couple of days which c an grow in numbers and restored to the normal morphology.4.To establish detection methods that detecting henzalkonium chloride by liquid chromatography-mass spectrometry and phenoxy ethanol by gas chromatography-mass spectrometry in biological samples:BAC’s linear conc--entration at the range of 0.800μg/L-200μg/L was good.The within day pre cision was less than 5%,while the recovery rate in method was more than 85%;phenoxyethanol’s linear concentration at the range of 0.05mg/mL～50mg/mL is good.The within day precision was less than 5%,while methods and extractions recovery rate is above 90%.5.The metabol ism experiment of borneol and preservatives in the conj--unctival sac:The metabolism time of BAC-12 and BAC-14 in the conjunctiv--al sac of t1/2 was 61.81h and 88.76h,the MRT was 63.24min and 106.39min.Borneol can significantly accelerate the metabolism of BAC in conjunctiv--al sac,and significantly prolong the time of benzalkonium chloride on ocular surface,it prompts that borneol can increase the accumulation of benzalkonium chloride on ocular surface.The time of Phenoxy ethanol in the conjunctival sac of t1,2 was 55.88h,MRT was 30.59min,borneol can sig--nificantly accelerate thephenoxy ethanol in the conjunctival sac of met--abolism,but does not extend phenoxy thanol in the dwell time of ocular surface.6.Borneol and preservatives to ocular surface injury:long--term deliv--ery of 0.1mg/mL benzalkonium chloride on ocular surface to big table in--jury,it can lead to dry eye,combined with Borneol can increase the resi--due in the cornea and sclera and aggravate the ocular surface injury.3mg/mL Phenoxy ethanol has mild nature,long-term administration of the ocular surface cause small injury and could not aggravate the injury.The damage of long-term use of 1mg/mL borneol on ocular surface is not big,but if apply.it to ophthalmology preparation,it still need to study its interaction with other drugs.Conclusion:Long-term drip into eyes with BAC,it can lead to big irritation on ocular surface,goblet cells decrease and dry eyes,if combinedwith Borne ol can increase the accumulation of benzalkonium chloride in ocular surf ace tissue,prolong the time of benzalkonium chloride on ocular surface and aggravate injury on ocular surface.Phenoxyethanol has mild nature,long-term administration of the ocular surface cause small injury and could not aggravate the injury.The damage of long-term use of lmg/mL borneol on ocular surface is not big,but if apply it to ophthalmology p reparation,it still need to study its interaction with other drugs.There fore,the need for long-term use of eye drops should not use benzalkonium chloride asa bacteriostatic agent,so as not to cause dry eye;avoid the use of BAC as a preservatives in eye drops containing borneol.,may give priority to using phenoxy ethanol.If select the benzalkonium chloride as bacteriostatic agent in Ophthalmic preparation,it should add eye prote--ction component to the prescription.