Mihoko N. Nakashima

Time course of nitric oxide synthase activity in neuronal, glial, and endothelial cells of rat striatum following focal cerebral ischemia.

Summary1. The time course of nitric oxide synthase (NOS) activity in neuronal, endothelial, and glial cells in the rat striatum after middle cerebral artery (MCA) occlusion and reperfusion was examined using a histochemical NADPH-diaphorase staining method.2. In sham-operated rats, neuronal cells of the striatum exhibited strong NADPH-diaphorase activities. When rats were subjected to MCA occlusion for 1 hr, neuronal damage, including neurons with positive NADPH-diaphorase activities, appeared in the striatum at 3 hr after and extended to all areas of the striatum 3–4 days after reperfusion.3. NADPH-diaphorase activities in the endothelial cells increased in the damaged part of striatum from 3 hr after, peaked at 1–2 days after MCA occlusion/reperfusion, then gradually decreased.4. In parallel with the development of neuronal damage, some astrocytes and a high proportion of microglia/macrophages located in the perisite and in the center of the damaged striatum, respectively, exhibited a moderate to high level of NADPH-diaphorase activities. Most of these activities disappeared at 4 days after MCA occlusion.5. These findings provided evidence that an inappropriate activation of NOS in endothelial cells and microglia/macrophages, in response to MCA occlusion/reperfusion, is closely associated with initiation and progression of ischemic neuronal injury in the striatum.

Simultaneous determination of arylpropionic acidic non-steroidal anti-inflammatory drugs in pharmaceutical formulations and human plasma by HPLC with UV detection

A simple and sensitive high-performance liquid chromatography-UV detection method was developed for the simultaneous determination of non-steroidal anti-inflammatory drugs (NSAIDs) having an arylpropionic acid moiety in pharmaceutical formulations and human plasma. Isocratic separation was employed on ODS column (250 x 4.6 mm i.d., 5 microm) at ambient temperature. The mobile phase consisted of acetonitrile, phosphate buffer (pH 3.5; 50 mM), methanol and tetrahydrofuran. The NSAIDs in the eluent were monitored under a wavelength-programme to provide their maximum absorbance. Mefenamic acid was used as an internal standard. Drugs were found to be 96.8-101.9% of their label claim in pharmaceutical formulations. One hundred microliters of human plasma samples were pretreated with a simple liquid-liquid extraction using ethyl acetate. The detection limits of compounds studied at a signal-to-noise ratio of 3 were 11.5-75 ng/ml in human plasma samples. The proposed method is simple, selective and could be applicable for routine analysis of arylpropionic acidic NSAIDs in pharmaceutical as well as in human plasma samples.

Fluorometric determination of dl-fenfluramine, dl-norfenfluramine and phentermine in plasma by achiral and chiral high-performance liquid chromatography

High-performance liquid chromatography (HPLC) with fluorescence detection has been developed for the simultaneous determination of sympathomimetic amines including ephedrine, norephedrine, 2-phenylethylamine, 4-bromo-2,5-dimethoxyphenylethylamine, phentermine (Phen) and DL-fenfluramine (Fen) in spiked human plasma. Furthermore, an enantioselective HPLC method for the separation of D-Fen (dexfenfluramine) and L-Fen (levofenfluramine) in addition to their active metabolites D- and L-norfenfluramine (Norf) is described. The detection was achieved at emission wavelength of 430 nm with excitation wavelength of 325 nm for both methods. The analytes were extracted from plasma (100 microl) at pH 10.6 with ethyl acetate using fluoxetine as the internal standard. The extracts were evaporated and derivatized with the fluorescence reagent 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride in the presence of carbonate buffer (pH 9.0). A gradient separation was achieved on a C18 column for the achiral separation or on a Chiralcel OD-R column for the chiral separation. The methods were fully validated, and shown to have excellent linearity, sensitivity and precision. The chiral method has been applied for the determination of D- and L-enantiomers of Fen and Norf, in addition to Phen in rat plasma after an intraperitoneal administration of DL-Fen and Phen, simultaneously.

Liquid chromatography studies on the pharmacokinetics of phentermine and fenfluramine in brain and blood microdialysates after intraperitoneal administration to rats.

A highly sensitive and simple HPLC method with fluorescence detection for the determination of phentermine (Phen), fenfluramine (Fen) and norfenfluramine (Norf, the active metabolite of Fen) in rat brain and blood microdialysates has been developed. The brain and blood microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) in the presence of carbonate buffer (0.1 M, pH 9.0) at room temperature. The chromatographic conditions consisted of an ODS column and mobile phase composition of acetonitrile and water (65:35, v/v) with flow rate set at 1.0 ml/min. The detection was performed at excitation and emission wavelengths of 325 and 430 nm, respectively. Under these conditions, the DIB-derivatives of Phen, Fen and Norf were well separated and showed good linearities in the studied ranges (5-2000 nM for Phen and 10-2000 nM for Norf and Fen) with correlation coefficients greater than 0.999. The obtained detection limits were less than 23 fmol on column (for the three compounds) in both brain and blood microdialysates at a signal-to-noise ratio of 3 (S/N=3). The intra- and the inter-assay precisions were lower than 10%. The method coupled with microdialysis was applied for a pharmacokinetic drug-drug interaction study of Phen and Fen following individual and combined intraperitoneal administration to rats. In addition, since the role of protein binding in drug interactions can be quite involved, the method was applied for the determination of total and free Phen and Fen in rat plasma and ultrafiltrate, respectively. The results showed that Fen and/or Norf significantly altered the pharmacokinetic parameters of Phen in both blood and brain but did not alter its protein binding. On the other hand, there was no significant difference in the pharmacokinetics of Fen when administered with Phen.

High performance liquid chromatography with fluorescence detection for the determination of phenylpropanolamine in human plasma and rat’s blood and brain microdialysates using DIB-Cl as a label

A high performance liquid chromatographic method for the determination of phenylpropanolamine (PPA) in human plasma and rats brain and blood microdialysates using fluorescence (FL) detection after precolumn derivatization with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) is described. PPA was extracted from plasma samples by a liquid-liquid extraction method with ethyl acetate followed by derivatization with DIB-Cl, while the blood and brain microdialysates were directly subjected for derivatization. The DIB-derivatives of PPA and the internal standard, ephedrine (EP), were then separated using an isocratic HPLC-FL set at excitation and emission wavelengths of 325 and 430 nm, respectively, on an ODS column. Calibration curves of PPA in spiked human plasma were linear over the concentration range of 5-5000 nM (0.755-755 ng/ml) and those in spiked blood and brain microdialysates were linear over the range of 25-5000 nM (3.775-755 ng/ml) with limits of detection of 17, 48 and 40 fmol on column in plasma and blood and brain microdialysates, respectively. As well, the intra- and the inter-assay precisions were lower than 12% for human plasma and the microdialysates. The method was successfully applied for the monitoring of PPA levels in rats brain and blood microdialysates administered with a single oral dose of PPA (2.5 mg/kg).

High-performance liquid chromatography with peroxyoxalate chemiluminescence determination of propentofylline concentrations in rat brain microdialysate

A high-performance liquid chromatographic determination of a neuronal cell protective compound, propentofylline [3-methyl-1-(5-oxohexyl)-7-propyl-7H-purine-2(3H),6(1H)-dione] was performed combining a microdialysis technique with peroxyoxalate chemiluminescence (PO-CL) detection. The microdialysate was subjected to a fluorescent derivatization of propentofylline with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H) without further cleanup, because the membrane used in the microdialysis excluded high-molecular-mass proteins. The proposed method showed a good linearity in the determination range of 0.031 to 1.25 ng/injection; y (microV)=4234 x (ng)- 13.43, r=0.9993 (y=peak height and x=amount of propentofylline). The very low determination limit of 0.031 ng/injection was ca. 200 times more sensitive than that of HPLC-UV determination. The HPLC-PO-CL method was applied for the first time to determine propentofylline concentration in the dialysate obtained from the rat hippocampus after a single oral administration (25 mg/kg). Propentofylline showed its maximum extracellular fluid (ECF) concentration of 125.1+/-15.1 ng/ml (mean+/-SD, n=3) at 0.33 h after administration.

Semi-micro column HPLC of triazolam in rat plasma and brain microdialysate and its application to drug interaction study with itraconazole.

Semi-micro column high-performance liquid chromatographic method with ultraviolet detection for the determination of triazolam (TZ) in rat plasma and brain microdialysate is described. The separation was achieved on a 250 x 1.5 mm, i.d. C(18) column and the column effluent was monitored at 222 nm. The detection limits at a signal-to-noise ratio of 3 obtained using spiked plasma and artificial cerebrospinal fluid were 2.1 and 0.7 ng/ml, respectively. The method was applied to drug-drug interaction study of TZ with itraconazole (ITZ). The peak concentration (C(max)) and the area under the curve (AUC) of TZ in brain microdialysate after simultaneous administration of TZ (2.5 mg/kg, intravenously (i.v.)) and ITZ (25 mg/kg, p.o.) to rats increased 3.4-folds (P<0.001) and 2.9-folds (P<0.001), respectively, compared to those of TZ alone. Also, the AUC of TZ in plasma increased 2.6-folds and remarkable delay in its elimination half-life (t(1/2)) was observed. The concentrations of TZ in brain microdialysate and plasma were also measured after single administration of TZ (2.5 mg/kg, i.v.) to rats pretreated with daily administration of ITZ (25 mg/kg, p.o.) once a day for a week. There was no significant difference in TZs C(max) in both ITZ treatments (P>0.2) however its t(1/2) after the daily pretreatment with ITZ was significantly increased (P<0.05). In plasma, the AUC of TZ after daily pretreatment of ITZ was lower than the single combined treatment, but significantly different from TZs AUC in the absence of ITZ (P<0.05). As a result, single simultaneous administration of TZ with ITZ and single administration of TZ after daily pretreatment with ITZ to rats, ITZ seriously interfered with the pharmacokinetic parameters of TZ in plasma and brain micodialysate.

Population pharmacokinetic investigation for optimization of amiodarone therapy in Japanese patients.

Background: Optimal use of amiodarone (AMD) requires information regarding the drugs pharmacokinetics and the influence of various factors on the drugs disposition. This study was conducted to establish the role of patient characteristic in estimating doses of AMD using nonlinear mixed effects modeling in Japanese patients treated with oral therapy. Methods: Serum concentrations of AMD were determined by high-performance liquid chromatography. The 151 serum trough concentrations from 23 patients receiving repetitive oral AMD were collected. Analysis of the pharmacokinetics of AMD was accomplished using a 1-compartment open pharmacokinetic model. The effect of a variety of developmental and demographic factors on AMD disposition was investigated. Results: Estimates generated by nonlinear mixed effects modeling indicated that the clearance of AMD was influenced by the demographic variables: total body weight (TBW), daily dosage of AMD (DD), body mass index (BMI), gender (GEN), duration of AMD dosing (DUR), and patient clearance factor (Conc&thetas;; Conc = serum trough concentration of AMD). The final pharmacokinetic parameters were CL/F (L/h) = 0.072·TBW·Conc−1.01·1.95DD≧200·0.931BMI≧25·1.37GEN·DUR−0.016, and Vd/F (L) = 78.4·TBW, where CL is total body clearance and Vd is volume of distribution. As all doses were given orally, it was impossible to assess the bioavailability (F). DD ≧200 is an indicator variable that has a value of 1 if the patient is receiving more than 200 mg daily dosage of AMD, and 0 otherwise. BMI ≧25 is an indicator variable that has a value of 1 if the BMI is 25 kg/m2 and over, and 0 otherwise. GEN is an indicator variable that has a value of 1 if the patient is woman, and 0 otherwise. Conclusions: The authors developed new population pharmacokinetic parameters. Clinical application of the findings in the present study to patient care may permit selection of an appropriate initial maintenance dose, thus enabling the clinician to achieve a desired therapeutic effect.

HPLC OF (±)-FENFLURAMINE AND PHENTERMINE IN PLASMA AFTER DERIVATIZATION WITH DANSYL CHLORIDE

A high-performance liquid chromatographic method is described for the simultaneous determination of the dansyl derivatives of (±)-fenfluramine (Fen) and phentermine (Phen) in addition to four other sympathomimetic amines, namely, norephedrine (NE), ephedrine (E), 2-phenylethylamine (2-PEA), and 4-bromo-2,5-dimethoxyphenethylamine (2-CB), using fluoxetine (FLX) as an internal standard. The separation was performed on a reversed-phase C18 column, using a gradient system and fluorescence detection. The dansylation conditions were examined and the optimum derivatization was obtained at pH 9.0 with 4.7 mM dansyl chloride concentration. A preliminary study for the simultaneous determination of the dansyl derivatives in spiked human plasma was performed. The derivatives were well separated within 45 min and showed good linearities from 0.005 to 2μM for E, Phen, and Fen, from 0.002 to 0.8 μM for 2-CB, and from 0.025 to 2 μM for NE and 2-PEA with detection limits ranging from 16 to 255 fmol on the column. Extraction recovery of the compounds was greater than 90%. The applicability of the method was studied in rat plasma following an intraperitoneal administration of Fen and Phen.

Investigation of Protective Effects of Sodium Hyaluronate Eyedrop Against Corneal Epithelial Disorders Using an Electrophysiological Method

PURPOSE The purpose of this study was to investigate the protective effects of sodium hyaluronate eyedrop against corneal epithelial disorders caused by antiglaucomatous eyedrops using an electrophysiological method. METHODS Three kinds of antiglaucomatous eyedrops, including benzalkonium chloride (BAC) as an ophthalmic preservative, a BAC-free antiglaucomatous eyedrop, and a sodium hyaluronate eyedrop, were used in this study. Eyedrops were applied to excised rabbit corneas, and the electrophysiological property of the cornea was monitored using an Ussing chamber with a turnover system that mimics human tear turnover. With this system, changes in transepithelial electrical resistance (TER) in the corneal surface were recorded. RESULTS The corneal TER after applying antiglaucomatous eyedrops tended to decrease concomitantly with increasing the concentration of the BAC included as a preservative. On the other hand, there was no significant change in the corneal TER for the initial 60 min after applying sodium hyaluronate eyedrop compared with those of the control. Moreover, the pretreatment with a sodium hyaluronate eyedrop reduced the extent of decrease in the corneal TER observed after application of antiglaucomatous eyedrops alone. CONCLUSION Those results indicate that a sodium hyaluronate eyedrop has the potential to protect the corneal surface from antiglaucomatous eyedrops, including BAC as an ophthalmic preservative.