Barri Thaer, Trtić-Petrović Tatjana, Karlsson Michael, Jönsson Jan Ake
Department of Analytical Chemistry, University of Lund, P.O. Box 124, SE-22100 Lund, Sweden.
J Pharm Biomed Anal. 2008 Sep 10;48(1):49-56. doi: 10.1016/j.jpba.2008.04.030. Epub 2008 May 8.
The technique equilibrium sampling through membrane (ESTM) was extended to measuring the free drug concentration in solutions of drug and protein. Bjerrum and Scatchard plots were employed for characterizing individual drug binding to pure human blood proteins. Four drugs were investigated as a model system: fluvoxamine and ropivacaine which dominantly bind to alpha-acid glycoprotein (AGP), and R,S-ibuprofen and S-ketoprofen which highly bind to human serum albumin (HSA). The level of drug binding to AGP and HSA relied on drug and protein concentrations. Bjerrum and Scatchard plots revealed high affinity constants (Ka) at low protein concentration. Both Bjerrum and Scatchard plots of fluvoxamine and ropivacaine binding to AGP showed one specific binding site (n1=1) with ropivacaine Ka value close to 5 times higher than the Ka of fluvoxamine at 22.9 microM AGP concentration. Bjerrum plots of ketoprofen and ibuprofen gave total number of binding sites or bound molecules of 6-7, which did not depend on the drug or protein concentration. Scatchard plots of ketoprofen and ibuprofen exhibited two binding sites (n1 and n2) at 0.15 microM and 0.75 microM HSA concentrations. On one hand, at 0.15 microM HSA, ketoprofen and ibuprofen were bound to site I at n1=1.2 and n1=1.0, respectively. However, at 0.75 microM HSA, ketoprofen and ibuprofen were bound to site I at n1=1.2 and n1=1.9, respectively. On the other hand, site II, at 0.15 microM HSA, interacted with ketoprofen and ibuprofen at n2=5.6 and 6.7, respectively. However, at 0.75 microM HSA, site II interacted with ketoprofen at n2=7.4 and ibuprofen at n2=6.2. It would be concluded that, upon mixing ketoprofen and ibuprofen in a HSA solution, a ketoprofen-ibuprofen interaction would most likely occur at site II in HSA.
膜平衡采样技术（ESTM）被扩展用于测量药物与蛋白质溶液中的游离药物浓度。采用比耶鲁姆（Bjerrum）图和斯卡查德（Scatchard）图来表征药物与纯人血蛋白质的个体结合情况。研究了四种药物作为模型系统：主要与α-酸性糖蛋白（AGP）结合的氟伏沙明和罗哌卡因，以及与人类血清白蛋白（HSA）高度结合的R,S-布洛芬和S-酮洛芬。药物与AGP和HSA的结合水平取决于药物和蛋白质的浓度。比耶鲁姆图和斯卡查德图显示在低蛋白浓度下具有高亲和力常数（Ka）。氟伏沙明和罗哌卡因与AGP结合的比耶鲁姆图和斯卡查德图均显示一个特异性结合位点（n1 = 1），在22.9 microM AGP浓度下，罗哌卡因的Ka值比氟伏沙明的Ka值高近5倍。酮洛芬和布洛芬的比耶鲁姆图给出的结合位点总数或结合分子数为6 - 7，这与药物或蛋白质浓度无关。酮洛芬和布洛芬的斯卡查德图在0.15 microM和0.75 microM HSA浓度下显示两个结合位点（n1和n2）。一方面，在0.15 microM HSA时，酮洛芬和布洛芬分别以n1 = 1.2和n1 = 1.0与位点I结合。然而，在0.75 microM HSA时，酮洛芬和布洛芬分别以n1 = 1.2和n1 = 1.9与位点I结合另一方面，在0.15 microM HSA时，位点II分别与酮洛芬和布洛芬以n2 = 5.6和6.7相互作用。然而，在0.75 microM HSA时，位点II与酮洛芬以n2 = 7.4相互作用，与布洛芬以n2 = 6.2相互作用。可以得出结论，在HSA溶液中混合酮洛芬和布洛芬时，酮洛芬 - 布洛芬相互作用最有可能发生在HSA的位点II。
