Tatsuya Kitade

Second derivative spectrophotometric determination of partition coefficients of phenothiazine derivatives between human erythrocyte ghost membranes and water

The absorption spectra of six phenothiazine derivatives, chlorpromazine, triflupromazine, promazine, promethazine, trifluoperazine and prochlorperazine, measured in the solutions containing various amounts of human erythrocyte ghosts (HEG) showed bathocromic shifts according to the amount of HEG. Due to the strong background signals caused by HEG, the baseline compensation was incomplete, even though the sample and the reference solutions contained the same amount of HEG, hence further spectral information could not be obtained. The second derivative spectra of these absorption spectra clearly showed the derivative isosbestic points, indicating that the residual background signal effects were entirely eliminated. The derivative intensity differences of the phenothiazines (DeltaD values) before and after the addition of HEG were measured at a specific wavelength. Using the DeltaD values, the partition coefficients (K(p)) of these drugs were calculated and obtained with R.S.D. of below 10 %. The fractions of partitioned phenothiazines calculated from the K(p) values agreed well with the experimental values. The results indicate that the derivative method can be applicable to the determination of partition coefficients of drugs to HEG without any separation procedures.

Determination of p-aminobenzoic acid in the presence of bovine serum albumin by room-temperature phosphorimetry on a poly(vinyl alcohol) substrate

Solid substrate room-temperature phosphorimetry on a poly(vinyl alcohol) (PVA) substrate was applied to the analysis of p-aminobenzoic acid (PABA) in the presence of bovine serum albumin (BSA). The buffer solution containing PABA and BSA was spotted on the PVA substrate and after sufficient drying, room-temperature phosphorescence (RTP) measurements were performed on both the front and the back side of the substrate. The BSA was separated on the front surface of the substrate and it emitted a negligibly weak RTP signal. Therefore, the RTP measurement of PABA on the reverse of the sample spotted side was not interfered by BSA, whereas, impurities in the BSA permeated into the PVA and emitted a weak RTP signal on the back side of the substrate. However, the RTP intensities of the impurities were weak enough as compared to that of PABA under the experimental conditions, so that their interference could be neglected. To improve the RTP intensity and reproducibility of PABA in the presence of BSA, a new substrate that consisted of Millipore-VS and PVA layer was prepared. The analytical figures of merit of this substrate obtained under the best experimental condition for maximum phosphorescence emission are a relative standard deviation of 6.0% (n=5) at a level of 2 nmol/spot and a linear calibration curve from 0.5 to 20 nmol/spot with a correlation coefficient of 0.996 for 25 data points.

Preparation of immobilized glucose oxidase membrane by the plasma polymerization technique

Abstract Glucose oxidase was immobilized on a Millipore (MP) filter by coating with plasma-polymerized propargyl alcohol. The resulting immobilized enzyme membrane was used as a glucose sensor. The properties as a glucose electrode system were evaluated by amperometric response with either the steady-state method or the reaction rate method. The response was proportional to concentrations of the glucose solution up to 2 m M and the sensitivity was dependent on the amount of GOD impregnated into the MP filter.

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.

Localization of glass particles in animal organs derived from cutting of glass ampoules before intravenous injections

Abstract Glass particles derived from the cutting of glass ampoules and suspended in parenteral solutions were introduced into mouse organs by intravenous injections in the tail vein. Organs were removed, thin sectioned, and plasma ashed on glass slides by means of glow-discharged oxygen to remove organic matrices which interfered with microscopic observation of glass particles. The ashed specimens were subsequently coated with a thin film of plasma-polymerized tetrafluoroethylene which suppressed the hygroscopicity of the specimens so that they became hydrophobic and at the same time permanently preserved the microstructures of the ashed specimens. Microscopic investigation of the glass particles retained in the ashed specimens supported size-dependent localization of the glass particles in different organs; i.e., large particles over 20 μm in diameter were mostly retained in the lung, while smaller particles around 5–10 μm in diameter were found in the liver and spleen, and occasionally in the kidney. A mesh-screen effect was therefore supposed along the route of blood circulation. No glass particle was found in the brain. Large numbers of extremely small glass particles less than 5 μm in diameter were not accounted for in all the organs investigated, but a wide diffusion in the whole body at very low density was presumed.

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.

Alpha7 nicotinic acetylcholine receptor-specific agonist DMXBA (GTS-21) attenuates Aβ accumulation through suppression of neuronal γ-secretase activity and promotion of microglial amyloid-β phagocytosis and ameliorates cognitive impairment in a mouse model of Alzheimer's disease

We previously demonstrated that stimulation of nicotinic acetylcholine receptors (nAChRs) increases amyloid-β (Aβ) phagocytosis in rat microglia and is closely associated with the decrease of brain Aβ and amelioration of memory dysfunction in a transgenic mouse model of Alzheimers disease (AD). Here, we examined the subtypes of nAChRs involved in these beneficial effects. In primary cultures of rat microglia, the α7 nAChR selective agonist 3-[(2,4-dimethoxy)benzylidene]-anabaseine dihydrochloride (DMXBA) promoted Aβ and fluorescent latex bead phagocytosis, whereas selective α7 nAChR antagonists suppressed the enhanced Aβ phagocytosis. In a transgenic mouse model of AD, administration of DMXBA attenuated brain Aβ burden and memory dysfunction. Moreover, DMXBA suppressed γ-secretase activity in solubilized fractions of human neuroblastoma cells and transgenic mouse brain. These results suggested that selective activation of α7 nAChRs promoted microglial Aβ phagocytosis and suppressed neuronal γ-secretase activity to contribute to the attenuation of the brain Aβ burden and cognitive impairment. Thus, we propose neuronal and microglial α7 nAChRs as new therapeutic targets in the treatment of AD.

Polydiacetylene Liposomal Aequorin Bioluminescent Device for Detection of Hydrophobic Compounds

In this study, a polydiacetylene liposomal aequorin bioluminescent device (PLABD) that functioned through control of the membrane transport of Ca(2+) ions was developed for detecting hydrophobic compounds. In the PLABD, aequorin was encapsulated in an internal water phase and a calcium ionophore (CI) was contained in a hydrophobic region. Membrane transport of Ca(2+) ions across the CI was suppressed by polymerization between diacetylene molecules. On addition of an analyte, the membrane transport of Ca(2+) ions across the CI increased, and Ca(2+) ions from the external water phase could diffuse into the internal water phase via the CI, which resulted in bioluminescence of the aequorin. Lidocaine, procaine, and procainamide were used as model compounds to test the validity of the detection mechanism of the PLABD. When each analyte was added to a suspension of the PLABD, bioluminescence from the aequorin in the PLABD was observed, and the level of this bioluminescence increased with increasing analyte concentration. There was a linear relationship between the logarithm of the analyte concentration and the bioluminescence for all analytes as follows: R = 0.89 from 10 nmol L(-1) to 10 mmol L(-1) for lidocaine, R = 0.66 from 10 nmol L(-1) to 100 μmol L(-1) for procaine, and R = 0.74 from 100 nmol L(-1) to 100 μmol L(-1) for procainamide. Compared to the traditional colorimetric method using polydiacetylene liposome, the PLABD was superior for both the sensitivity and dynamic range. Thus, PLABD is a valid, simple, and sensitive signal generator for detection of hydrophobic compounds that interact with PLABD membranes.

Effect of Long-Chain Fatty Acids on the Binding of Triflupromazine to Human Serum Albumin: A Spectrophotometric Study

Human serum albumin (HSA) in the blood binds long-chain fatty acids (LCFAs), and the number of bound LCFAs varies from 1 to 7 depending on the physical condition of the body. In this study, the influence of LCFA-HSA binding on drug-HSA binding was studied using triflupromazine (TFZ), a psychotropic phenothiazine drug, in a buffer (0.1 M NaCl, pH 7.40, 37°C) by a second-derivative spectrophotometric method which can suppress the residual background signal effects of HSA observed in the absorption spectra. The examined LCFAs were caprylic acid (CPA), lauric acid (LRA), oleic acid (OLA), and linoleic acid (LNA), respectively. Using the derivative intensity change of TFZ induced by the addition of HSA containing LCFA, the binding mode of TFZ was predicted to be a partition-like nonspecific binding. The binding constant (K M−1) showed an increase according to the LCFA content in HSA for LRA, OLA, and LNA up to an LCFA/HSA molar ratio of 3–4. However, at higher ratios the K value decreased, i.e. for OLA and LNA, at an LCFA/HSA ratio of 6–7, the K value decreased to 40% of the value for HSA alone. In contrast, CPA, having the shortest chain length (8 carbons) among the studied LCFAs, induced a 20% decrease in the K value regardless of its content in HSA. Since the pharmacological activity of a drug is closely related to the unbound drug concentration in the blood, the results of the present study are pharmaco-kinetically, pharmacologically, and clinically very important.