| Drug protein bingding rate is the important pharmacological parameters of drugin animals,which affect the drug’s free concentration and the disposal of drugs invivo.It has made a great development in mammal.However,little information isavailable about drug binding with plasma protein on aquacultuer animal. In thisstudy,We use the equilibrium dialysis and ultrafiltration method to determine theplasma protein binding rate of difloxacin（DIF） in vitro and vivo.We study the bindingproperties of DIF binding to plasma protein in Carassaisauratus gibebioin vitro.Wedesignthe Aeromonashy drophila-infected group and the untreated as control toanalyze the binding properties of DIF binding to plasma protein of Carassaisauratusgibebio in vivo.We analyze the correlation between animal physiology,plasma proteinbinding and pharmacokinetics.The major results were summarized as followings:1.Established the method to assay the binding ratio of DIF to plasma protein ofCarassaisauratus gibebio,inspected the condition of equilibrium dialysis andultrafiltration to seprate the free drug concentration and bond drugconcentration,compared the plasma protein binding rate evaluate by HPLC （highperformance liquid chromatography）,equilibrium dialysis and ultrafiltration.The resultshowed that the adsorption of dialysis bag and ulrtafiltration membrane discern lessthan6.45±0.051%and4.51±0.077%;The equilibrium time of dialysis was48h,theultrafine filter completely separated in the centrifugal condition of12000g,120min.The recovery rate of drug in blood plasma and dialysate （ultrafiltrate）were all higher than92.5%and97.0%respectively;The within-day precision were allless than0.99%and0.48%，inter-day precision all less than0.84%and0.90%;Thebinding rate of DIF by equilibrium dialysis and ultrafiltration was19.6-32.4%and19.24-30.01%respectively.The result showed the equilibrium dialysis can used tostudy drug plasma protein binding property in vitro,the ultrafiltration can used tostudy drug plasma protein binding character in vivo.2.Using equilibrium dialysis method,we detected the binding rate of plasma protein toDIF in vitro.We investigated the effects of temperature, concentrations of plasma andthe drug on the rates on the binding characters of DIF to plasma protein in Carassaisauratus gibebio.The results showed that the binding rates were not higherthan that of the temperature at4,15,20,25,37℃,while the binding rate of plasma wassignificant higher than that of double（8.87±0.39%） and quarter（4.09±0.24%）dilutedplasma（p>0.05）.The values of the binding rates were decreased as the drugconcentration raising,which found to be32.4-19.6%when the concentration of thedrug ranged from0.5μg/mL to5μg/mL;while the blinding rate of plasma protein tothe drug has no difference when the concentration was5-40μg/mL.The biggestcombining ability （β） and the dissociation constant （Kdp） were3.5x10-7mol/g and4.04x10-6mol/L respectively.In addition, the value of the dissociated drug has a linearrelationship with the total concentration of the drug,which equation isy=0.77x-0.18.The results indicated that the blinding rates of plasma protein inCarassaisauratus gibebio to DIF are drug concentration dependent,which are notaffected by temperature under the experiment condition,and shows a linearrelationship when the concentration of DIF ranges from5-40μg/mL.However,Thereis nonlinear relationship at0.5-5μg/mL.3.Studying the binding properties of DIF binding to plasma protein ofCarassaisauratus gibebio in vivo.Investigating the diversities of plasma proteinbinding rate in infected group and healthy group and analyzing the relationshipbetween total and free drug concentration and the regularity ofbinding-dissociation.The results showed that the binding rate in infected group higherthan healthy group.Excluding the ALB decreased significantly,theTP,ALT,AST,γ-GT,CREA and UREA all increased significantly in infected group.Theplasma protein binding rates had logarithmic relationships with the total drugconcentration and their equqtions were y=-9.01lnx+74.34（infected group）andy=-4.81lnx+65.15（healthy group）,while the equations between the plasma proteinbinding rates and free concentration of the drug were y=-6.36lnx+64.91（infectedgroup）and y=-4.36lnx+60.63（healthy group）in these two groups.The biggestcombining ability（Ka）and the dissociation constant（Kdp）were3.1×10-7mol/g and3.1×10-7mol/L respectively in infected group and4.2×10-7mol/g and9.6×10-7mol/L.The results indicate that the values of the the plasma protein bindingrates of DIF could be increased by Aeromonashy drophila in Crucian Carp,DIFbinding with plasma protein are also drug concentration dependent and have the lowerdissociation in vivo.4. Objective to investigate the relationship between the diversities of plasma protein binding rate of DIF and its pharmacokinetic in Carassaisauratus gibebio,Determining the vivo plasma protein binding rate of change of DIF,analyzing thepharmacokinetic characteristics and the plasma protein binding rate of DIF. Theresults showed that the plasma protein binding rates of the drug in infected groupand control group was negatively correlated features with the free drugconcentration;Plasma drug concentration-time curve of infected group andcontrol group both can be described by open two compartments model,DIFabsorption and elimination of infected group were slower than that of the controlgroup, the apparent volume of distribution and area under curve were bigger thanthat of control group;The elimination of infected group’s free drug was fasterthan that of control group,the apparent volume of distribution and area undercurve was less than that of control group. The results displayed that the increasesof Plasma protein binding rate leading to the drug storage in the blood by drugcombination, which may cause drug distribution limited, slow elimination andprolonged retention in the blood, therefore, we should reduce drug （DIF） useinterval, increase the volume of drug use to achieve the goal of prevention andtreatment in the clinical treatment. |
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