Binding of diuretic antihypertensive bendroflumethiazide to human serum albumin studied by ¹9F nuclear magnetic resonance method.

Simultaneous specific and nonspecific binding of bendroflumethiazide (BFZ) to human serum albumin (HSA) and concentration profile of BFZ in HSA buffer (pH 7.40) solution were investigated by ¹9F nuclear magnetic resonance (NMR) method. The ¹9F NMR spectrum of BFZ (200 µM) in a buffer solution showed a sharp signal of its CF3 group at 17.8 ppm from the reference trifluoroethanol. Addition of 0.60mM HSA to the sample solution caused the CF(3) signal splitting into three broadened peaks at 18.4 (A), 17.9 (B) and 17.4 ppm (C). By its chemical shift and spectral behavior, B was assigned to unbound BFZ. Competition experiments with Site I and II ligands lead to C being assigned to Site II bound BFZ. However, the peak intensity (areas) of A was not reduced by these ligands, suggesting that A arises from nonspecific binding. Using the peak intensities at several total concentrations of BFZ, Scatchard plot was performed. The plot for A provided a straight line parallel to the x-axis confirming nonspecific binding and that for C was consistent with specific binding. The binding constants for nonspecific and specific Site II binding were 1.02 and 1.00 × 104 (M⁻¹) (n=1.1), respectively. The presence of 0.10 M Cl⁻ in the sample solution affected the binding constant of Site II binding, but not that of nonspecific binding. The concentration profile of BFZ calculated using the binding constants revealed that nonspecific binding is more effective than Site II binding for the binding of BFZ to HSA. It was also confirmed that considerable amounts of BFZ liberated from Site II by the Site II ligands or Cl⁻ ions bind again nonspecifically.

Determination of partition coefficients of diazepam and flurazepam between phosphatidylcholine bilayer vesicles and water by second derivative spectrophotometric method.

Second derivative spectrophotometry allowed the establishment of a simple and accurate method for the determination of partition coefficients of benzodiazepine drugs in a liposome/water system. The absorption spectra of diazepam (DZ) and flurazepam (FZ) in phosphatidylcholine (egg yolk) bilayer vesicle suspensions showed small spectral changes depending on the concentration of phosphatidylcholine vesicles. However, the intense background signals caused by the light scattering of the phosphatidylcholine vesicles made it difficult to yield a correct base line, thus the quantitative spectral data could not be obtained. In the second derivative spectra, the spectral changes were enhanced and three derivative isosbestic points were observed for each drug indicating the entire elimination of the residual background signal effects. The derivative intensity change of each drug (DeltaD) induced by its interaction with phosphatidylcholine bilayers was measured at a specific wavelength. From the relationship between the DeltaD value and the lipid concentration, the molar partition coefficients (K(p)s) of DZ and FZ were calculated and obtained with a good precision of R.S.D below 10%. The fractions of the partitioned DZ and FZ calculated by using the obtained K(p) values agreed well with the experimental values. The results prove that the derivative method can be usefully and easily applied to the determination of partition coefficients of benzodiazepines in the liposomes/water system without any separation procedures.