Study On Drug-Human Serum Albumin Interaction By Capillary Electrophoresis

An important topic in the drug discovery and development is the study of drug-serum albumin binding. The methods for the studying interaction of doxycycline, eight fluoroquinolones as well as 5-fluorouracil and human serum albumin (HSA) were established by application of capillary electrophoresis technique. The results show that the thermodynamic parameters and interaction forces can be estimation by capillary electrophoresis-frontal analysis (CE-FA) method for the binding of drug and protein. This study expands the application of CE-FA for studying the drug-protein binding. The main content of this paper is as follows:Chapter?:Review. Drug-protein interaction research methods, HSA structural characteristics, the quantitative determination of drug-protein binding parameters by capillary electrophoresis, the applications of capillary electrophoresis in drug-protein interaction studies were introduced.Chapter?:The interaction of doxycycline and HSA. The binding of doxycycline to HSA, under simulated physiological conditions (pH 7.4,67 mM phosphate,I=0.17, drug concentration 100?M, HSA concentration up to 475?M,36.5?) was studied by CE-FA method. The number of primary binding sites, binding constant and physiological protein binding percentage were 1.9,1.51×103 M-1 and 59.80%, respectively. In addition, the thermodynamic parameters including enthalpy change (AH), entropy change (?S) and free energy change (AG) of reaction were obtained in order to characterize the acting forces between doxycycline and HSA. Furthermore, to better understand the nature of doxycycline-HSA binding and to get information about potential interaction with other drugs, displacement experiments were performed. The results showed that doxycycline binds at "site?" of HSA.Chapter?:Interaction of fluoroquinolones and HSA. The binding of fluoroquinolones to the transport protein, HSA, under simulated physiological conditions has been studied by CE-FA methods. The binding of these drugs to human plasma was evaluated by using ultrafiltration and capillary electrophoresis. The free drug concentration [D]f at each HSA concentration was determined by the plateau height in the electropherograms and the calibration lines. The binding constants of fluoroquinolones and HSA were estimated using nonlinear regression with origin 7.5 software. The fluoroquinolones were found to show low affinity toward HSA, with binding constants ranging from 1.73×102 to 5.40×102 M-1. The percentages of protein binding for fluoroquinolones to HSA were between 8.6 and 22.2%, while the protein binding percentages for fluoroquinolones to human plasma were between 10.2 and 33.1%. It can be found that the protein binding percentages for fluoroquinolones to HSA are mostly lower than that for fluoroquinolones to human plasma. It suggests that HSA is the primary protein responsible for the binding of fluoroquinolones in human plasma. The thermodynamic parameters were obtained by CE-FA. The positive?H and?S values obtained by CE-FA showed that the binding reaction was an endothermic process, and hydrophobic interaction played major roles in the binding of fluoroquinolones to HSA.Chapter IV:The interaction of 5-fluorouracil and HSA. First, the binding of 5-fluorouracil to HSA was studied by CE-FA. Because of the similar electrophoretic mobility, 5-fluorouracil and HSA could not be separated and could not got the drug plateau peak under simulated physiological conditions (pH 7.4,67 mM phosphate,1=0.17,36.5?). So we investigated the effect of Amylose, dextrin, dextran,?-cyclodextrin (?-CD), methyl-?-cyclodextrin (Me-P-CD), hydroxypropyl-?-cyclodextrin (HP-?-CD) and sulfobutyl ether?-cyclodextrin (SBE-?-CD) to the separation of the peaks. The results showed that the concentration of unbound 5-fluorouracil could be determined accurately when 50 mM Me-?-CD or over 40 mM HP-?-CD was added to the buffer, though the two peaks could not be separated completely. We studied the binding of 5-fluorouracil and HSA, used 50 mM HP-?-CD as buffer additive. It showed that the binding of 5-fluorouracil and HSA was relatively weak, and It could not get the number of binding sites and binding constant. Then, the binding of 5-fluorouracil to HSA was studied by affinity capillary electrophoresis (ACE). The binding constant was 9.19×104 M-1 (36.5?). The thermodynamic parameters including enthalpy change (?H), entropy change (?S) and free energy change (?G) of reaction were obtained in order to characterize the acting forces between 5-fluorouracil and HSA. In addition, to better understand the nature of 5-fluorouracil-HSA binding and to get information about potential interaction with other drugs, displacement experiments were performed. The results showed that 5-fluorouracil binds HSA at "site I".Chaper?:Conclusion. The application of interaction of doxycycline, fluoroquinolone, 5-fluorouracil and HSA by CE were summaried.