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.

Effects of Particle Size and Cholesterol Content on the Partition Coefficients of Chlorpromazine and Triflupromazine between Phosphatidylcholine-Cholesterol Bilayers of Unilamellar Vesicles and Water Studied by Second-Derivative Spectrophotometry.

Phosphatidylcholine(PC)-cholesterol (0-30 mol%) unilamellar vesicles of several sizes (20-600 nm) were prepared in buffer (pH 7.4) solutions by sonication or extrusion methods. The vesicle size was measured by a dynamic light-scattering method. Absorption spectra of chlorpromazine (CPZ) and triflupromazine (TFZ) in the presence of these vesicles showed a bathochromic shift according to the increase in vesicle concentration, but the counterbalance of the baseline was incomplete due to the intensive light scattering by the vesicles; thus, no isosbestic point could be observed. In the second-derivative spectra, the residual background signal effects were eliminated and three derivative isosbestic points were clearly observed for both drugs. The derivative intensity change (DeltaD) induced by the addition of the vesicles was measured at the lambda(max) of each drug. From the relationship between the DeltaD value and the lipid concentration, the partition coefficients (K(p)) of CPZ and TFZ between these vesicles and water (buffer) were calculated. The results revealed that the vesicle size (20-600 nm) and preparation method do not affect the K(p) values, and although the incorporation of cholesterol into the PC bilayers induces a decrease of the K(p) values, the vesicle size also did not affect the K(p) values in vesicles of the same cholesterol content. Copyright 1999 Academic Press.

Determination of the partition coefficient of triflupromazine between phosphatidylcholine vesicles and water by (19)F nuclear magnetic resonance spin-lattice relaxation time measurement.

The (19)F nuclear magnetic resonance (NMR) spin-lattice relaxation time (T(1)) of the trifluoromethyl signal of triflupromazine (TFZ) was measured in aqueous suspensions of phosphatidylcholine (lecithin) small unilamellar vesicles (SUV). The observed T(1) value depended on the concentration of SUV. Based on a simple two-site rapid exchange model, the partition coefficient (K(p)) of TFZ between lecithin SUV and water was calculated from the relationship between the T(1) value and the lecithin concentration by using a nonlinear least-squares method. The obtained K(p) value (2.1+/-0.2x10(5)) agreed well with that measured by a second-derivative spectrophotometric method. The (19)F NMR T(1) method will be useful for the determination of partition coefficients of drugs having fluorine atom(s), especially for the drugs which do not have absorption in the ultraviolet or visible region, or for those having absorption but do not show any changes according to their incorporation into the lecithin bilayers. The method does not require any separation procedure that may disturb the equilibrium conditions.