Atsuhiro Mizuno

Inhibitory Effect of MCI-186, a Free Radical Scavenger, on Cerebral Ischemia Following Rat Middle Cerebral Artery Occlusion

1. In this study, we investigated the effect of a radical scavenger, MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one), on cerebral damage induced by rat middle cerebral artery (MCA) occlusion, and further measured the hydroxyl radical level at the ischemic border zone using a microdialysis technique. 2. MCI-186, at a dose of 3 mg/kg per 30 min, was administered as a continuous infusion two times for 30 min, starting 20 min and then 80 min after Rose Bengal injection. 3. MCI-186 significantly (P < 0.05) reduced size of cerebral damage 24 hr after MCA occlusion and significantly (P < 0.05) reduced hydroxyl radical level.

Analysis of nicotine content of hair for assessing individual cigarette-smoking behavior.

Summary: The concentration of nicotine in hair was measured by a capillary gas chromatograph with a nitrogen-phosphorus detector after hair was dissolved in 2.5 N sodium hydroxide and nicotine was extracted using diethyl ether. In hair samples obtained from 36 smokers, who had smoked ≥3 years, there was a significant positive correlation between the concentration of nicotine and the number of cigarettes consumed daily (r = 0.685; p < 0.001). The nicotine content of white hair was much less than that of black hair collected from the same subjects with grizzled hair. The above findings were confirmed by an animal experiment, in which nicotine (0.2, 0.6, and 2 mg/kg/day for 2 weeks) was administered intraperitoneally to pigmented rats whose hair had been removed beforehand by plucking from an area of the back of each rat. The hair which grew back in the denuded area was plucked and collected one week after the last administration of nicotine. The nicotine concentration in the brown hair of the back correlated with the given dose (r = 0.824; p < 0.001). In a separate experiment, hairs were removed from areas of back and thorax, and nicotine delivered to the rats for 4 weeks by an osmotic pump implanted under the skin, showing that the concentration in the whitish hair was much lower than that in brown hair. Finally, hair samples were collected from 14 subjects who had participated in a smoking cessation program over 6 months and who abstained from cigarette smoking. The axial distribution of nicotine along the hair shafts was determined for each subject. The month-by-month decrease in the self-reported number of consumed cigarettes agreed roughly with the cm-by-cm decrease in nicotine content along the hair shafts. The data suggest that analysis of nicotine along the hair shafts may serve as a useful way of assessing individual cigarette-smoking behavior over a period from several months to a number of years.

Dihydropyrimidine Dehydrogenase Activity in 150 Healthy Japanese Volunteers and Identification of Novel Mutations

Purpose: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme catalyzing the metabolic degradation of the anticancer drug 5-fluorouracil (5-FU). Population studies of DPD activity in peripheral blood mononuclear cells (PBMC) were reported in healthy volunteers and cancer patients. Although these studies were done in mainly Caucasian and African American populations, only a little information is available for a Japanese population. Experimental Design: One hundred fifty healthy Japanese volunteers were screened for a population distribution of PBMC-DPD activity. Genetic analysis of a volunteer with very low DPD activity was carried out by reverse transcriptase-PCR and genomic sequencing. Bacterially expressed recombinant mutant DPD proteins were purified and characterized. Results: Mean and median values of PBMC-DPD activity for 5-FU reduction in the study population were 0.173 and 0.166 nmol/min/mg protein, respectively. A 57-year-old female volunteer (proband in this study) had very low DPD activity (0.014 nmol/min/mg protein) with a very low level of expression of DPD protein. Two novel nucleotide substitutions, at nucleotide positions 1097 (1097G > C) and 2303 (2303C > A), resulting in amino acid substitutions at positions 366 (G366A) and 768 (T768K), respectively, were identified. The G366A mutation caused not only a marked decrease in the affinity of the enzyme to cofactor NADPH but also reduced Vmax for 5-FU-reducing activity to ∼0.5. T768K mutant lost its activity much faster than did wild DPD. Conclusions: We found one healthy volunteer (0.7% of the population) with very low PBMC-DPD activity due to heterozygosity for a mutant allele of the DPYD gene in a population of 150 Japanese.

Inhibitory effect of MS-153 on elevated brain glutamate level induced by rat middle cerebral artery occlusion.

BACKGROUND AND PURPOSE In this study we investigated the effects of a novel compound, MS-153 ([R]-[-]-5-methyl-1-nicotinoyl-2-pyrazoline), on elevated brain glutamate concentrations and cerebral infarct volume induced by middle cerebral artery (MCA) occlusion in the rat. METHODS The rat MCA was occluded by a thrombus induced by a photochemical reaction between green light and the photosensitizer dye rose bengal, which causes endothelial injury followed by formation of a platelet- and fibrin-rich thrombus at the site of photochemical reaction; this method is routinely used in our laboratory to produce arterial occlusion in experimental animals. Extracellular glutamate concentration at the ischemic border zone was determined by a microdialysis technique. The size of cerebral infarction was measured by a histochemical technique 24 hours after MCA occlusion. MS-153 was administered at various doses as a continuous infusion for 24 hours, beginning 0 to 2 hours after MCA occlusion. RESULTS At the ischemic border zone, the concentration of glutamate in the extracellular fluid increased by 40-fold after ischemia. At 3.13 mg/kg per hour, MS-153 reduced glutamate concentration (P < .05) and also the size of ischemic cerebral infarction (P < .05). Furthermore, the glutamate uptake inhibitor DL-threo-beta-hydroxyaspartate reversed the effect of MS-153 on glutamate concentration. CONCLUSIONS The reduction in the size of cerebral infarction by MS-153 may be attributable to the inhibition of glutamate release or an increase in cellular glutamate uptake.

Simultaneous determination of ofloxacin, norfloxacin and ciprofloxacin in human hair by high-performance liquid chromatography and fluorescence detection

A high-performance liquid chromatographic method for the simultaneous determination of ofloxacin, norfloxacin and ciprofloxacin in human hair is described. A reversed-phase C18 column and a fluorescence detector with switching fluorescence wavelengths were used together with solid-phase extraction of the drugs from hair dissolved in 1 M sodium hydroxide. Reproducibility and linearity studies yielded coefficients of variation of 0.2-2.2, 1.4-3.1 and 1.5-3.4%, and correlation coefficients of 1.000, 0.999 and 0.999 within the concentration range 0.3-100 ng/ml for ofloxacin, norfloxacin and ciprofloxacin, respectively. For validation, hair samples were obtained from six subjects who had been taking one or two of the three fluoroquinolones. Assuming a hair growth-rate of 1 cm per month fluoroquinolones could be detected in the hair section(s) that had grown approximately between the dates of drug administration and hair sampling.

Effect of 21-aminosteroid lipid peroxidation inhibitor, U74006F, in the rat middle cerebral artery occlusion model.

The aim of this study was to evaluate the effect of 21-aminosteroid lipid peroxidation inhibitor, U74006F, on ischaemic brain tissue damage using the rat middle cerebral artery occlusion model. Under anaesthesia, the left middle cerebral artery was exposed without cutting the dura mater via a subtemporal craniotomy, under an operating microscope. Photo-illumination (wave length; 540 nm) was applied to the middle cerebral artery and then rose bengal (20 mg/kg) was administered intravenously. The middle cerebral artery was completely occluded by thrombus about 6 min after the administration of rose bengal. U74006F (1.0 mg/kg) was then injected intravenously just after the cessation of illumination. Twenty four hours after the operation, the extent of ischaemic damage was measured by magnetic resonance imaging technique. After measuring the extent of ischaemic damage, the brain was immediately removed from animals treated with or without U74006F for determination of lipid peroxidation, and the generation of free arachidonic acid in the brain. U74006F significantly (P < 0.01) reduced the size of ischaemic damage. Twenty-four hours after the operation, lipid peroxidation and the concentration of free arachidonic acid in the left hemisphere (infarction side) were significantly (P < 0.05) higher than in the right hemisphere. U74006F significantly (P < 0.05) decreased the content of lipid peroxidation products and free arachidonic acid. There was a significant (P < 0.05) correlation between the extent of ischaemic damage and the concentration of lipid peroxidation products in the left hemisphere 24 h after the operation. In conclusion, U74006F might reduce the extent of ischaemic damage by inhibiting lipid peroxidation in the brain, thus minimizing oxidative damage to neural tissues.

Ofloxacin in human hair determined by high performance liquid chromatography.

A procedure is presented for quantitating ofloxacin (OFLX) in human scalp hair by high performance liquid chromatography (HPLC) with a fluorescence detector. An octadecylsilane (ODS) column was used and the mobile phase was a mixture of potassium phosphate buffer (pH 2.6) and acetonitrile. The recovery of OFLX was 90.9-93.8% and within- and between-run precisions were 0.35-1.41% and 1.41-5.49% as the coefficient of variation (CV), respectively, when 5-50 ng OFLX was added to 1 mg blank hair. The calibration curve was linear in the range of 0.5-50 ng/tube (0.5 ml). Interference with other quinolone derivatives could be avoided according to the difference in their retention times or fluorescence spectra. Several pieces of hair were obtained from each of twelve healthy male volunteers, who had taken OFLX (100, 300 or 900 mg total dose) over a 1-3 day period 2 weeks before the hair sampling. In all hair samples except one obtained from a volunteer, who had taken the lowest dose, the 2-cm long segments nearest the scalp contained OFLX (5-45 ng/mg hair), while the upper segments did not. A highly significant positive correlation was observed between the total dose and the concentration of OFLX in the 2-cm long hair segments. Such a positive correlation was also revealed in rat hair sampled after repeated i.p. administration of OFLX over a 5-week period. These results suggest that the measurement of OFLX in hair by the present method would be useful for testing patient compliance in clinical pharmacology as well as for application to forensic science.

Evaluation of a GPIIb/IIIa antagonist YM337 in a primate model of middle cerebral artery thrombosis.

We compared the antithrombotic effect of anti-GPIIb/IIIa antibody Fab fragment YM337 with that of a thromboxane A2 synthetase inhibitor, sodium ozagrel. With the monkeys under halothane anesthesia, the right middle cerebral artery was observed via a transorbital approach without cutting the dura mater. Photoillumination (wavelength 540 nm) was applied to the middle cerebral artery, and then rose bengal (20 mg kg(-1)) was administered intravenously. The experimental drugs were intravenously injected 15 min before rose bengal injection and followed by continuous infusion for 3 h after dye injection. The thrombotic occlusion induced by this photochemical reaction in monkey middle cerebral artery was reproducible. YM337 significantly prolonged the time to first occlusion and the total time of arterial patency during the 3-h observation period after dye injection. In contrast, sodium ozagrel had no significant effect. YM337 but not sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation. However, while sodium ozagrel significantly inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Neurological deficit in the YM337-treated animals was significantly milder than that in the control group. The area of infarct in the YM337 treatment animals was smaller than that in the control group. The novel selective GPIIb/IIIa antagonist YM337 was effective in ameliorating the decrease in patency of the middle cerebral artery and reducing the area of cerebral infarction in monkeys.

Phase I Clinical Studies of 7432-S, a New Oral Cephalosporin: Safety and Pharmacokinetics

Phase I clinical studies of 7432‐S, a new oral cephalosporin, including a randomized placebo‐controlled trial were conducted with 40 healthy volunteers, in single‐dose studies, 7432‐S was orally administered at doses of 25, 50, 100, and 200 mg. The mean plasma levels peaked at 2.1 to 3.0 hours and reached 1.9, 3.6, 5.6, and 11.6 μg/ml, respectively. Linear correlation was observed between plasma AUC values and doses given. The half‐lives of the plasma levels were 0.88 to 2.26 hours with a mean of 1.53 ± 0.33 hours. The mean urinary recoveries were 67.5 to 75.2% of the dose within 24 hours. 7432‐S was partially metabolized to 7432‐S‐trans which was excreted in urine at 7.2 to 9.2% of the doses. Study of the meal effect showed that AUC values and peak levels were not altered although the time to the peak levels was slightly prolonged, In multiple‐dose studies, 100 mg of 7432‐S twice daily for 2 weeks and 200 mg twice daily for 1 week were administered and there was no abnormal accumulation of 7432‐S in plasma throughout the study. No significant differences were observed in plasma levels and urinary recoveries between single‐ and multiple‐dose regimens. Clinical symptoms, physical tests, laboratory parameters, and fecal levels of vitamins K1 and K2 were in normal ranges. 7432‐S was concluded to be safe and well tolerated.

Pharmacokinetics and safety of l-carnitine infused i.v. in healthy subjects.

SummaryThe pharmacokinetics and safety of a brief i. v. infusion of l-carnitine 0, 20, 40 and 60 mg/kg have been investigated in 10 healthy subjects.The diurnal intraindividual variability of plasma carnitine was small (C. V.=3.0, 3.9 and 3.9%, respectively), and the total 24 h excretion in urine was also small and relatively constant: 4.6, 21.5 and 13.0 mg/day in the controls vs 4.6, 20.2 and 6.0 mg/day during treatment in the three subjects to whom saline alone was administered according to a single-blind design. Therefore, the pre-dose level of carnitine was subtracted from the level after dosing for the pharmacokinetic analysis. Plasma carnitine fitted well to a three-compartment open model, with Vc of 0.11–0.20 l/kg and a t1/2γ of 10–23 h. The urine recovery in 24 h was 77.2–95.4%.There were no objective or subjective side-effects attributable to carnitine, so its i. v. infusion is considered to be safe.