Study On Preparation And Characterization Of Itraconazole Loaded Solid Lipid Nanoparticle For Ocular Topical Application And Ocular Pharmacokinetics In Rabbits

Background:Itraconazole?ITZ?is a new generation of triazole broad-spectrum antifungal drugs,which is effective against infections such as Candida,Cryptococcus neoformans and Aspergillus.ITZ is hydrophilic and poorly soluble in water.At present,itraconazole is mainly available as oral solid preparations?tablets and capsules?and injections.These preparations may relatively cause severe liver toxicity in clinical use,and the concentration of the drug reaching the eye tissues is relatively low,so it is needed to develop an ocular drug delivery system for the topical administration to achieve the therapeutic level.Solid lipid nanoparticles?SLNs?that composed of biocompatible and biodegradable materials are colloidal nanoparticulate systems designed and developed to deliver lipophilic drugs.These particulates are in the nanometer size range.The application of itraconazole loaded solid lipid nanoparticles?ITZ-SLNs?in the eye has not been reported.The general idea designed in this paper is to improve the permeability into cornea and ocular bioavailability of lipophilic itraconazole in the eye by developing ITZ-SLNs after topical administration.Purpose:The purpose of this experiment was to prepare an ophthalmic itraconazole loaded solid lipid nanoparticle formulation,evaluate its physicochemical properties,safety,and investigate its ocular pharmacokinetics?PK?in rabbit eyes.Methods:The microemulsion and low-temperature cool down solidification method was used to prepare itraconazole solid lipid nanoparticles?ITZ-SLN?.Capryol 90 and Precirol ATO 5 were used as the oil phase,Kolliphor?HS 15,glycerol and water were used as the water phase.The particle size,encapsulation efficiency,and drug loading capacity were used as evaluation indicators,the central composite design-response surface method was used to optimize the formulation of ITZ-SLN.The appropriate concentration of plant gel was added to the ITZ-SLN solution to improve the stability of the preparation.High performance liquid phase?HPLC?method was established to detect the content of the drug.ITZ-SLN was characterized by the particle size,zeta potential and polydispersity index?PDI?using Nanometer particle size analyzer?Nano ZS-90?.The encapsulation efficiency of ITZ in nanoparticles was determined by reverse HPLC.Diffusion-ultrafiltration method was used to evaluate the in vitro release behavior of ITZ-SLN.Ocular irritation of ITZ-SLN was evaluated on the healthy rabbit eyes without eye disease One single dose of 100?L of 0.1%ITZ-SLN eye drop was applied in the right conjunctival sac,and the same dose of normal saline in the left eye as a control.The clinical and slit lamp observations were performed at 1h,2h,4h,24h,48h and 72h after administration,and the score was evaluated according to the Draize scoring rules.ITZ levels in tears fluids,cornea and aqueous humors of rabbits were determined by HPLC.Ocular pharmacokinetics of ITZ-SLN was investigated after one single dose topical administration in New Zealand white rabbits.The forty-two animals were randomly divided into experimental group and control group,the two groups were further randomly divided into seven subgroups according to administration time points,respectively.Both eyes of the rabbits in the experimental group are given one single dose of 50?L of 0.1%ITZ-SLN while each eye of the control group received a single administration of 50?L of 0.1%ITZ-Susp,respectively.The tear fluid samples were taken at 5min,15min,30min,60min,90min,120min,and 180min after administration,and the animals were sacrificed,aqueous humor was aspirated using 1ml-syringe with 26G needle and the corneal tissues and were dissected.The ITZ concentrations in the samples were measured by HPLC-UV method.Another 42 New Zealand white rabbits were taken and randomly divided into different subgroups same as the rabbits with intact cornea epithelium.The administration was started 2 hours after the diameter of 6.25mm corneal epithelium was removed.The tear fluid samples were taken at 5min?15min?30min?60min?90min?120min?180min after the administration,the animals were sacrificed,the aqueous humor and the corneal tissues samples were taken.The concentration of ITZ in the samples was detected by HPLC-UV method.All data were statistically conducted.PK parameters were performed by DAS 2.1 and Graphpad prism 8.4 software.Results:The optimized formulation of ITZ-SLN was:drug:lipid=1:40,lipid:surfactant=1:4.5,surfactant:cosurfactant=2:1.The relationship between ITZ peak area and concentration showed good linearity in the range of 0.625-10?g/ml,the relative standard deviation?RSD?of the intra-day and inter-day were less than 10%.The average particle size of ITZ-SLN is?15.0±2.1?nm,polydispersity index?PDI?is0.135±0.02,zeta potential is?-22.65±0.91?m V,drug encapsulation efficiency is?96±2.1?%,the loading rate is?0.15±0.02?%.The in vitro release meets with the first-order release kinetics equation.There was no significant difference between the experimental group and the control group?P>0.05?in the irritation test,thus the results showed that the ITZ-SLN had no obvious irritation to rabbit eyes.The RSD of the drug in tears,cornea and aqueous humor for inter and intra-days were all<10%,and the recoveries were in the range of 85%to 115%.For the rabbits with intact epithelium,the peak time(T_max)of ITZ in the cornea was 15 minutes and the maximum concentration(C_max)was?7.92±1.29??g/g,the half-life(t_1/2)was 30.66min in the experimental group.The T_max of ITZ in the cornea was 5min,and the C_maxof ITZ in cornea was?3.08±0.99??g/g in the control group.The drug levels in cornea at each predetermined time points in the experimental group were significantly higher than in the control group at corresponding time points?P<0.05?.The T_max of ITZ in the tears of the experimental group and the control group were 5min,and the peak concentrations were?737.01±93.38??g/g and?477.64±20.8??g/g,the t_1/2 were 20.40min and 16.58min,respectively.The drug levels in tears at each predetermined time points in the experimental group were significantly higher than in the control group at corresponding time points?P<0.05?.For the rabbits with the deepithelium cornea,the T_max of ITZ in the cornea was 15 minutes,the C_maxof ITZ in cornea was?10.78±1.35??g/g,and the t_1/2 was 33.84min in the experimental group.The T_max of ITZ in the cornea was 5 minutes,and the C_maxof ITZ in cornea was?5.80±0.88??g/g,the t_1/2 in the cornea was 26.37min in the control group.The drug levels in cornea at 5min and 15 min after administration in the experimental group were significantly higher than in the control group at corresponding time points?P<0.05?,and there was no significant difference at the other corresponding time points..The T_max of ITZ in the tears in the experimental group and the control group was 5 minutes,and the C_max was?641.37±30.88??g/g and?404.18±46.14??g/g,the t_1/2 was 21.57min and 12.83min,respectively.The drug levels in tears at each predetermined time points in the experimental group were significantly higher than in the control group at corresponding time points?P<0.05?.For the rabbits with intact epithelium,the area under the concentrations-time curves(AUC_0-180min)in the cornea for the experimental group was 608.85?g/g·min,The AUC_0-180min in cornea of the control group were 60.75?g/g·min.The AUC _0-180min in cornea of the experimental group was 10.02times of the control group.The AUC_0-180min in tears of the experimental and control group were 28945.82?g/g·min and 13792.59?g/g·min,respectively.The AUC_0-180min in tears of the experimental was 2.1times of the control group.For the rabbits with the deepithelium cornea,the corneal AUC _0-180min of the experimental group was 724.63?g/g·min.the AUC_0-180min of the control group was 138.27?g/g·min,the corneal AUC_0-180min of the experimental group was 5.24 times of the control group,and the AUC_0-180minin tears of the experimental group and the control group were 23334.29?g/g·min and 1536.43?g/g·min,respectively.and the AUC_0-180min in the tears of the experimental group was 2.02 times of the control group,.The concentrations of the ITZ in the aqueous humor for rabbits with the intact and deepithelium corneal were below the detection limit.Conclusion:The ocular drug delivery system of ITZ-SLN was convenient to be prepared,ultra-small particle size and not irritant.It can effectively improve the permeability into the cornea and increase the ocular bioavailability of drugs after topical adminstration.