Biopharmacy, Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland.
J Pharm Biomed Anal. 2012 Dec;71:219-27. doi: 10.1016/j.jpba.2012.08.009. Epub 2012 Aug 17.
We evaluated fluorescence anisotropy measurements as an alternative technique to estimate drug-lipid membrane partitioning of fluorescent solutes.
The lipid bilayer partitioning of six drugs (logP in octanol/water between 2.6 and 5.4) was investigated by fluorescence anisotropy measurements with egg phosphatidylcholine liposomes at pH 7.4. Anisotropy was measured at about 5 and 50μM drug and varying lipid concentrations between 3 and 700μM. Fluorescence was corrected for light scattering and membrane affinities were estimated by non-linear regression analysis of the relative anisotropy as a function of lipid and solute concentrations. Liposome partitioning was in addition determined by equilibrium dialysis and potentiometric titration for comparison.
Correction for light scattering by the liposomes was possible to some extent for two drugs. The estimated partition coefficients of three drugs were concentration-independent. For two drugs, membrane saturation was expected at the higher drug concentration. One drug showed significant differences between the parameters estimated at high and low drug concentrations, indicating measurement artifacts. Linear regression between the estimated logarithmic partition coefficients from anisotropy measurements and equilibrium dialysis revealed a slope of 1.05 and an intercept of 1.16 (n=5, r(2)=0.87).
Anisotropy measurement with liposomes is hampered by light scattering and pH-dependent fluorescence properties of ionizable drugs. Taking these limitations into account, the technique may offer an alternative to established methods for the estimation of drug membrane partitioning, in particular when potentiometric titration or equilibrium dialysis are not applicable.
我们评估荧光各向异性测量作为替代技术，以估计荧光溶质与药物-脂质膜的分配。
通过荧光各向异性测量，在 pH 7.4 下用卵磷酯脂质体研究了六种药物（在辛醇/水中的 logP 在 2.6 至 5.4 之间）的脂质双层分配。在约 5 和 50μM 药物和 3 至 700μM 之间的变化脂质浓度下测量各向异性。校正了光散射的荧光，并通过非线性回归分析相对各向异性作为脂质和溶质浓度的函数来估计膜亲和力。此外，通过平衡透析和电位滴定法进行脂质体分配的测定，以进行比较。
对于两种药物，通过脂质体进行光散射的校正在一定程度上是可能的。三种药物的估计分配系数与浓度无关。对于两种药物，预计在较高药物浓度下会出现膜饱和。一种药物在高和低药物浓度下估计的参数之间存在显著差异，表明存在测量伪影。从各向异性测量和平衡透析中估计的对数分配系数之间的线性回归显示斜率为 1.05，截距为 1.16（n=5，r²=0.87）。
脂质体的各向异性测量受到光散射和可电离药物的 pH 依赖性荧光性质的阻碍。考虑到这些限制，该技术可能为估计药物膜分配提供替代方法，特别是当无法进行电位滴定或平衡透析时。
