Shigehiko Takegami

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.

Differential effects of calcium antagonists on ABCG2/BCRP-mediated drug resistance and transport in SN-38-resistant HeLa cells

The effects of 9 calcium antagonists on ABCG2/BCRP-mediated resistance and transport were examined in HeLa and SN-38-resistant HeLa (HeLa/SN100) cells, overexpressing ABCG2/BCRP. Sensitivity to mitoxantrone, an ABCG2/BCRP substrate, in HeLa/SN100 cells was significantly reversed by the coexistence of the calcium antagonists, except for diltiazem and verapamil. The accelerated transport activity of Hoechst33342, an ABCG2/BCRP substrate, in HeLa/SN100 cells was significantly decreased by the presence of the calcium antagonists, except for diltiazem, nifedipine or verapamil, returning to the level of HeLa cells. The present study classifies the calcium antagonists into 3 categories: strong (benidipine, felodipine, nicardipine, nisoldipine and nitrendipine), moderate (amlodipine and nifedipine) and weak (diltiazem and verapamil) inhibitors of ABCG2/BCRP.

19F NMR spectrometric determination of the partition coefficients of some fluorinated psychotropic drugs between phosphatidylcholine bilayer vesicles and water

A simple 19F NMR spectrometric method was proposed for the determination of the partition coefficients of fluorinated psychotropic drugs, trifluoperazine (TFPZ), flunitrazepam (FNZ) and flurazepam (FZ) between phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUVs) and water (buffer). Each 19F NMR spectrum of these drugs in the presence of PC SUV showed a single signal accompanying a PC concentration-depending shift change and broadening, which indicated a fast exchange of these drugs between the water phase and the PC bilayer of SUV. From the relationship between the 19F chemical shift change (Deltadelta) of each drug and the PC concentration, the molar partition coefficients (K(p)s) were calculated and obtained with a good precision of RSD below 6%. The fractions of the partitioned drugs calculated by using the obtained K(p)-values were in a good agreement with the experimental values. The results demonstrate that the 19F NMR method can be usefully applied to the determination of partition coefficients of many drugs having fluorine atom(s) without any separation procedure, especially for drugs which do not have absorption in the ultraviolet or visible region, or those having absorption but show insignificant spectral changes according to their incorporation to PC bilayers (e.g. FNZ).

Synthesis of [N‐13CH3] drugs (chlorpromazine, triflupromazine and promazine)

These [N-13CH3] containing drugs (chlorpromazine, triflupromazine and promazine) have been synthesized by means of a two-step demethylation/methylation procedure. Copyright

Application of 19F NMR Spectroscopy Using a Novel a-Tocopherol Derivativeas a 19F NMR Probe for a Pharmacokinetic Study of Lipid Nano-Emulsionsin Mice

Objective: The aim of our study was to demonstrate the usefulness of 19F nuclear magnetic resonance (NMR) spectroscopy as an analytical technique applicable for the pharmacokinetic studies of lipid nano-emulsions (LNEs) using a mixture of soybean oil, phosphatidylcholine and sodium palmitate as drug carriers. Methods: A α−tocopherol derivative, 19F-TP, in which a 4-(trifluoromethyl) benzoyl group was introduced to the hydroxyl group of α-tocopherol was newly synthesized as a 19F NMR probe. Three different LNEs containing 19F-TP, denoted 19F-TP-LNEs (Small-LNE, Large-LNE, and polyethylene glycol-modified LNE (PEG-LNE)) were prepared by the sonication method and characterized using a dynamic light-scattering method and zeta potential analysis. The concentrations of the three 19F-TP-LNEs in the blood, liver and kidneys of mice were periodically evaluated based on the 19F NMR signal intensity ratio of 19F-TP using 0.1 mM of trifluoromethane sulfonic acid sodium salt as an internal reference. Results: 19F-TP was easily synthesized with a high yield of 96% in a one-step procedure. Small-LNE, Large- LNE and PEG-LNE had the mean particle sizes of 58, 157 and 174 nm and zeta potentials of –34, –53 and –32 mV, respectively. A single signal attributable to 19F-TP in 19F-TP-LNEs was observed at 15.4 ppm in the 19F NMR spectra of biological samples, but was observed to decrease over time. From the change of 19F NMR signal of 19F-TP in biological samples, it was shown that three 19F-TP-LNEs had different pharmacokinetic characteristics because of their droplet sizes and surface physical properties. Conclusion: Based on these results, the 19F NMR method was confirmed to be a convenient and useful tool for assessing the pharmacokinetics of LNEs without the need for complicated pretreatment procedures such as the deproteination of the matrix and extraction of the target compound before the 19F NMR measurements.

Determination of the partition coefficient of triflupromazine between phosphatidylcholine vesicles and water by 19F 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.

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.

Characterization, in vitro cytotoxicity and cellular accumulation of paclitaxel-loaded lipid nano-emulsions

Lipid nano-emulsions (LNEs) having a mean droplet size of ∼50 nm were investigated as drug carriers for paclitaxel (TXL) to achieve its satisfactory loadings and to develop a pharmaceutically acceptable alternative to the current formulation, Taxol®. TXL was incorporated into the LNEs at 2.0 mg/ml without changes in particle size or drug precipitation. In the cytotoxicity study, TXL-loaded LNEs had cytotoxicity to HeLa cells equivalent to that of TXL alone; the 50% growth inhibitory concentrations (IC50) of TXL-loaded LNEs and TXL alone were 1.53 ± 0.23 nM and 1.76 ± 0.08 nM, respectively. However, a cellular accumulation study using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a fluorescent probe showed that the accumulation of DPH-loaded LNEs in HeLa cells was remarkably lower than that of DPH alone. These results indicated that LNEs were a useful vehicle for TXL, even though LNEs themselves could not be efficiently accumulated in HeLa cells.

Electrochemiluminescence Bioassays with a Water‐Soluble Luminol Derivative Can Outperform Fluorescence Assays

The most efficient and commonly used electrochemiluminescence (ECL) emitters are luminol, [Ru(bpy)3 ]2+ , and derivatives thereof. Luminol stands out due to its low excitation potential, but applications are limited by its insolubility under physiological conditions. The water-soluble m-carboxy luminol was synthesized in 15 % yield and exhibited high solubility under physiological conditions and afforded a four-fold ECL signal increase (vs. luminol). Entrapment in DNA-tagged liposomes enabled a DNA assay with a detection limit of 3.2 pmol L-1 , which is 150 times lower than the corresponding fluorescence approach. This remarkable sensitivity gain and the low excitation potential establish m-carboxy luminol as a superior ECL probe with direct relevance to chemiluminescence and enzymatic bioanalytical approaches.

Partitioning of organophosphorus pesticides into phosphatidylcholine small unilamellar vesicles studied by second-derivative spectrophotometry

In order to quantitatively examine the lipophilicity of the widely used organophosphorus pesticides (OPs) chlorfenvinphos (CFVP), chlorpyrifos-methyl (CPFM), diazinon (DZN), fenitrothion (FNT), fenthion (FT), isofenphos (IFP), profenofos (PFF) and pyraclofos (PCF), their partition coefficient (Kp) values between phosphatidylcholine (PC) small unilamellar vesicles (SUVs) and water (liposome-water system) were determined by second-derivative spectrophotometry. The second-derivative spectra of these OPs in the presence of PC SUV showed a bathochromic shift according to the increase in PC concentration and distinct derivative isosbestic points, demonstrating the complete elimination of the residual background signal effects that were observed in the absorption spectra. The Kp values were calculated from the second-derivative intensity change induced by addition of PC SUV and obtained with a good precision of R.S.D. below 10%. The Kp values were in the order of CPFM>FT>PFF>PCF>IFP>CFVP>FNT⩾DZN and did not show a linear correlation relationship with the reported partition coefficients obtained using an n-octanol-water system (R(2)=0.530). Also, the results quantitatively clarified the effect of chemical-group substitution in OPs on their lipophilicity. Since the partition coefficient for the liposome-water system is more effective for modeling the quantitative structure-activity relationship than that for the n-octanol-water system, the obtained results are toxicologically important for estimating the accumulation of these OPs in human cell membranes.