| Text：Objective: To study the photodynamic activity of liposomal Hypo-crellin B(HB) on pulmonary microvascular endothelial cells of mice(PMVECS)and experimental choroidal neovascularization (CNV)， the pharmacokineticparameters of Liposomal HB in mice and the skin photoxicity of Liposomal HB inmice.Methods:1.The study of ptototoxiticy and dark-toxicity on PMVECS.In ptotogroup, thePMVECS were incubated with liposomal HB in vitro in different concentrations for4hours, and then irradiated by copper vapor laser with single wavelength578.2nm laserunder saturated light dose. In control group, the cells were incubated with liposomalHB in vitro in different concentrations for24hours only. The cell survival rate wasmeasured by MTT assay after24hours’incubation respectively. According to cellsurvival curves, the equation of each group was plotted. Finally IC50(50%inhibitionconcentration) of each group was derived. The IC50of liposomal HB was comparedwith HB.2.24BN rats were randomly divided into6groups according to the time afterptoto-coagulation3d,7d,14d,21d,28d and56d. Krypton laser photocoagulation wasperformed on the retina of experiment ental rats to set up a CNV animal model. Thewavelength was647nm, the spot size was50μm, the power was260mw, and theexposure time was200ms.There were8photocoagulation points in every rat eye. FFAand HE histology of CNV were analyzed at3d,7d,14d,21d,28d and56d afterphotocoagulation.3.20BN rats with experimental CNV were divided into blank control and PDTgroups. The PDT groups were made up of group A, B, C and D. The rats of group Aand B were injected with liposomal HB at1mg/kg. The rats of group C and D were injected with liposomal HB at2mg/kg. PDT was performed using light at awavelength of578.2nm, an power density of400mW/cm2and energy density of36J/cm2. The effect of these treatments on these structures was assessed by FFA andhistology.4.72Mice were assigned to12time groups randomly, receiving tail veininjection of Liposomal HB at dosages of2mg/kg body weight. The serum drugconcention at different time point was measured by fluorometric method. The serumdrug concention-time curve and the pharmacokinetic parameters were calculated withDAS ver2.1.1practical pharmacokinetics program.5.200Kunming mice were assigned to four dose groups randomly, each groupreceiving tail vein injection of0.9%NS at0.1ml/10g body weight, liposomal HB atdosages of1,2and4mg/kg body weight respectively. After a single tail vein injection,mice were exposed to simulated sun-light(40mW/cm2) for60minutes at1d,3d,5d,7d and14d. Skin phototoxicity was investigated by the weight of the slices taken fromearflaps by perforex(diameter=6mm).The back exposed skin, ear skin, liver andkidney of mice were sampled to observe the pathomorphology changes.Results:1.The IC50of liposomal HB was53.45ng/mL in PDT group, while the IC50ofliposomal HB was17.58μg/mL in dark group, which was3300fold of PDTgroup.There was not any dramatic difference about PDT IC50between liposomal HBand HB. But the dark IC50of liposomal HB was87fold of HB in simplephotosensitizers.2. FFA showed retinal edema after3d. Histology showed a great deal CNV wasformed after21d.The state of CNV reached stable at21d.3. The percentage of CNV that didn’t close in experimental groups wasdramatically low compared with controls(p＜0.01). The CNV closure at dose2mg/Kgwas superior than1mg/Kg. But at the same time, the damage to nomal retina andchroid in group2mg/Kg was classified as grade5, while the damage to nomal retinaand chroid in group1mg/Kg was classified as grade2.4.The mean serum drug concentration-time curve of liposomal HB after tail vein injection of a single dose (2mg/kg) in mice could be fitted to first order absorptionthree-compartment model with a weight of1. The main pharmacokinetic parameterswere: t1/2z=(2.319±0.462)h, Tmax=(0.127±0.048)h, max=(51.105±2.869) mg/Lrespectively.5. There were no significant difference of weights of ears between liposomalHB group(1mg/kg,2mg/kg and4kg/mg)and control group. Only2mice(liposomalHB,4kg/mg) showed light inflammatory reactions on the ear skins in1d. Others didnot show any obvious abnomalities.Conclusions:1.The photodynamic activity of liposomal HB on PMVECS was dramatic. Theliposomal HB is more safe than HB.2.The treatment with liposomal HB in the experimental CNV of rats waseffective. Under the unvarying illumination, the CNV closure varied as a function ofthe liposomal HB dose. At the same time, the damage to nomal retina and chroidvaried with the dose of liposomal HB too. Further research should be conducted tofind suitabie illumination and suitable dose.3. The drug distributed mainly in blood plasma and eliminated fairly quickafter administration, which corresponded to the features of ideal photosensitizer.4. liposomal HB has no obvious skin phototoxicity. |
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