M. Nakashima

Evaluation of the combination of a tissue-type plasminogen activator, SUN9216, and a thromboxane A2 receptor antagonist, vapiprost, in a rat middle cerebral artery thrombosis model.

Background and Purpose We aimed to evaluate a modified tissue-type plasminogen activator, SUN9216, and the combination of SUN9216 and a thromboxane A2 receptor antagonist, vapiprost, in a rat middle cerebral artery thrombosis model. Methods Under anesthesia, the left middle cerebral artery was observed under an operation microscope without cutting the dura mater via a subtemporal craniotomy. Photoillumination (wave length, 540 nm) was applied to the middle cerebral artery, and then rose bengal (20 mg/kg) was administered intravenously. The reopening of the middle cerebral artery by SUN9216, injected 30 minutes after middle cerebral artery occlusion, was observed under an operation microscope for a 60-minute observation period. Twenty-four hours after the operation, sections of the cerebrum were stained with triphenyltetrazolium chloride, and the area of cerebral infarction was analyzed by a computer. Results The combination of SUN9216 and vapiprost caused reopening of the middle cerebral artery in 58.8% of the rats, which was a greater percentage than that achieved with SUN9216 alone (31.6%). In contrast, saline did not cause reopening of the middle cerebral artery during the 60-minute observation period. The area of cerebral infarction in rats reperfused with SUN9216 was significantly reduced compared with that in the control group. The infarction area in rats treated with the combination of SUN9216 and vapiprost was reduced compared with that in rats treated with SUN9216 alone; this was the case whether or not the occlusion was reperfused. There was a significant correlation between the time of reopening of the middle cerebral artery and area of cerebral infarction. Conclusions A single injection of SUN9216 was effective in recanalizing the vessel and reducing the area of cerebral infarction.

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.

A new model for photochemically induced thrombosis in the inner ear microcirculation and the use of hearing loss as a measure for microcirculatory disorders

SummaryA new photochemical method was employed to cause disorders in the inner ears microcirculation, using the rat as an animal model. Hearing loss was used as a measure for establishing the altered microcirculation. Under pentobarbital anesthesia, the middle ear was opened by a ventral approach. The lateral wall of the cochlea was then illuminated with a filtered xenon lamp (wavelength 540 nm) while rose bengal was infused intravenously. Photoactivated rose bengal produces oxygen radicals and oxygen singlets, which subsequently damage the vascular epithelium to cause the adhesion and aggregation of platelets in the small vessels. Disintegration of the inner ear hair cells at the irradiated site became evident 24 h after the illumination. These findings further suggest that the photochemical occlusion in the inner ears microcirculation led to ischemic damage of the stria vascularis and the hair cells in the inner ear. When the action potential (AP) of the cochlea was measured with an electrocochleogram a gradual decrease occurred after the illumination. When acetylsalicylic acid was injected intravenously before treatment, the time required to completely suppress the AP was prolonged in a dose-dependent manner. Findings indicate that our method causes a photochemically induced occlusion in the inner ears microcirculation and is therefore potentially useful for evaluating the various effects of drugs on the ear.

An animal model for hearing disturbance due to inner ear ischemia: photochemically induced thrombotic occlusion of the rat anterior inferior cerebellar artery

SummaryA photochemical reaction between intravenous rose bengal and xenon light was used to induce a selective thrombus in the rat anterior inferior cerebellar artery (RICA). Compound action potentials (CAPs) were recorded by electrocochleography and cochlear blood flow (CBF) was monitored by laser Doppler flow-metry. Photothrombotic occlusion of the AICA caused inner ear ischemia to various degrees with or without alterations of the CAP. With use of this model we investigated the critical range of the CBF for preserving cochlear function, represented by the CAPs induced with 8 kHz half-wave of sinusoid at 100 dB SPL. Results then showed that a CBF range between 26.7% and 42.9% of baseline was somewhat critical for maintenance of cochlear function in an acute phase of ischemia. Pretreatment with heparin significantly delayed thrombotic occlusion of the AICA in a dose-dependent manner. Further use of our model for inner ear ischemia may be useful for studying pathophysiology and pharmacological therapy of cochlear disturbances subsequent to circulatory disorders.

Impairment of phagocytic cell respiratory burst by UVA in the presence of fluoroquinolones: an oxygen-dependent phototoxic damage to cell surface microvilli

Fluoroquinolones are widely used clinically as broad-spectrum antimicrobial agents. One of their side effects is UVA-dependent photosensitivity, observed after the skin is exposed to sunlight. We have investigated five fluoroquinolones and have found that their phototoxicity is oxygen dependent. Human phagocytic leucocytes were stimulated with serum opsonized zymosan to produce superoxide radical (O2-) (respiratory burst) in the presence of a sensitive O2(-)-specific cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazol (1,2-alpha) pyrazin-one hydrochloride (MCLA), as chemiluminescence reagent with which O2- can react to induce photon emission. The photon count was used as a measure of respiratory burst activity. When leucocytes were irradiated with UVA for 10 min in the presence of 3 micrograms ml-1 lomefloxacin, ciprofloxacin or norfloxacin, a marked decrease in respiratory burst activity was observed; in this respect, ofloxacin and tosufloxacin were weak. Scanning electron microscopy revealed that the cell surface microvilli were destroyed. The phototoxicity of fluoroquinolones could be abolished if oxygen in the tests was replaced by nitrogen or if the aminothiol DL-cysteine (1.5 mg ml-1) was added prior to irradiation. It is suggested that an oxygen species derived from UVA-excited drug molecules and oxygen mediates the phototoxicity of these fluoroquinolones.

Antiarrhythmic and electrophysiological effects of SD-3212, a novel Na+ and Ca++ channel blocker, in anaesthetized dogs with myocardial infarction in comparison with its stereoisomer (SD-3211) and bepridil

SummaryAntiarrhythmic and electrophysiological effects of SD-3212, a novel antiarrhythmic agent, which has both Na+ channel and Ca++ channel blocking activites, were compared with those of its (+)-stereoisomer, SD-3211, which has only a Ca++ channel blocking activity, and bepridil, a known Ca++ channel blocker with additional Na+ channel blocking activity, using the two-stage coronary ligation induced arrhythmia (24h after the ligation of the left anterior descending coronary artery) and 7 day-old myocardial infarcted hearts in anaesthetized dogs.SD-3212 showed a dose-dependent antiarrhythmic effect on the two-stage coronary ligation induced arrhythmia. SD-3212 at a dose of 3 mg/kg reduced the arrhythmic ratio, i.e. ectopic beats per min divided by the sum of ectopic beats and sinus beats per min, significantly from 1 up to 12 min after the administration. Neither bepridil (1–6 mg/kg) nor SD-3211 (1 mg/kg) had an antiarrhythmic effect. SD-3212 (0.3–3 mg/kg) prolonged both the conduction time in the normal myocardium and the delayed potential in the infarcted myocardium in the 7 day-old myocardial infarcted hearts in anaesthetized dogs in a dose-dependent manner. This effect of SD-3212 was shown at coupling intervals of 150–1000 ms increasing with decreasing interval. In this respect, SD-3212 is similar to drugs which show fast recovery of Vmax from use-dependent block such as lidocaine. Bepidril (1–6 mg/kg) also prolonged these parameters in a dose-dependent manner, however, the prolongation induced by bedripil was limited to shorter coupling intervals as compared with that induced by SD-3212. SD-3212 (0.1–1 mg/kg) did not show this prolonging effect. SD-3212 increased the refractory period in the infarcted zone to a small extent (not significantly) at all strengths tested between 0.5–4 mA and also in the normal zone between 0.2–1 mA to an even lesser extent than in the infarcted zone. Bepridil produced a significant increase of refractory period in the infarcted zone. In the normal zone, bepridil produced a non-significant, but greater increase of the refractory period as compared with SD-3212. SD-3211 did not affect the refractory period in the infarcted zone or in the normal zone. None of the three drugs produced a significant change in the excitation threshold.Thus, SD-3212 showed electrophysiological properties of a drug with fast recovery kinetics without producing a significant increase of refractory period, and these properties are very similar to those of class Ib antiarrhythmic agents such as lidocaine. The present study suggests that there might be a possibility of SD-3212 to become a safe and unique antiarrhythmic agent with suppresses both Na+ and Ca++ inward current.

Efficacy of a Low-Dose Omeprazole-Based Triple-Therapy Regimen for Helicobacter pylori Eradication Independent of Cytochrome P450 Genotype : The Japanese MACH Study.

AbstractObjectives: To investigate the efficacies of two different triple-therapy regimens (standard versus low doses), and the influence of cytochrome P450 enzyme (CYP) genetic polymorphism on these efficacies, in Japanese patients undergoing Helicobacter pylori eradication treatment. Methods: All patients received 1 week of triple therapy. Patients in group A (low-dose regimen) received omeprazole 40 mg/day + amoxicillin 1500 mg/day + clarithromycin 800 mg/day; patients in group B (standard-dose regimen) received omeprazole 40 mg/day + amoxicillin 2000 mg/day + clarithromycin 1000 mg/day. Results: A total of 225 patients (113 in group A and 112 in group B) were randomised to one of the two triple-therapy regimens. The eradication rates were 78.8% (89/113 patients; 95% CI 70.1, 85.9) in group A and 83.0% (93/112 patients; 95% CI 74.8, 89.5) in group B. Genetic polymorphism of CYP2C19, a major metabolic enzyme of omeprazole, did not affect eradication rates, while susceptibility to clarithromycin greatly affected the success of eradication. The cumulative ulcer relapse rate at 24 weeks after endoscopically documented ulcer healing (30 weeks after completion of the drug regimen) was 8.3% for group A and 12.5% for group B (log rank test: p = 0.6248). However, comparison of the cumulative relapse rate of 6.7% in patients after successful H. pylori eradication with the relapse rate of 27.3% in those who failed H. pylori eradication revealed a significant difference in the remission-time curve (log rank test: p = 0.0047). This finding suggested the existence of a relationship between H. pylori eradication failure and ulcer relapse. Both drug regimens were well tolerated. Endoscopically proven reflux esophagitis developed in about 10% of patients after eradication, but was not clinically significant. Conclusions: One week of triple therapy with a low-dose regimen provides adequate H. pylori eradication in Japanese patients. CYP genetic polymorphism is of minimal clinical significance with both triple-therapy regimens.

Effect of a Ca2+ entry blocker, nilvadipine, on hearing disturbances and equilibrium dysfunction caused by microcirculatory disorders of the rat inner ear

We evaluated the effects of Ca2+ entry blockers, nilvadipine and flunarizine, on microcirculatory disorders of the inner ear and on blood flow in the inner ear of rats. Under sodium pentobarbital anesthesia, the middle ear was opened by a ventrolateral approach. A green light (wave length 540 nm) was applied to the cochlea or the vestibule to induce a hearing disturbance or equilibrium dysfunction as a result of inner ear microcirculatory disorders, while rose bengal solution was infused intravenously. In a hearing disturbance model, a compound cochlear nerve action potential was recorded by electrocochleography every minute after the beginning of illumination. The sound stimulus was an 8 kHz sine wave 100 dB normal hearing level. The action potential was calculated 128 times. The action potential disappeared about 12 min after the beginning of illumination. In another model of equilibrium dysfunction, the photoillumination was applied for 40 min under the infusion of rose bengal. The behavior of rats was observed in the swimming test and nystagmus was recorded 24 h after the completion of photoillumination. In a separate experiment, blood flow in the inner ear was measured with a laser Doppler flowmeter under sodium pentobarbital anesthesia. In this study, both nilvadipine and flunarizine prolonged the time required for complete suppression of the action potential, prevented equilibrium dysfunction in the swimming test and reduced the occurrence of nystagmus. Flunarizine significantly increased inner ear blood flow and nilvadipine failed to decrease blood flow in the inner ear, despite a reduced systemic blood pressure. In conclusion, Ca2+ entry blockers may prevent microcirculatory disorders of the inner ear in rats.

A new model of equilibrium dysfunction in the rat induced by photochemical damage to the inner ear's microcirculation.

SummaryA new photochemical method was employed to damage the inner ear microcirculation in the rat. Under pentobarbital anesthesia, the middle ear was exposed by a ventral approach and the tympanic membrane and the malleus and incus were removed. The vestibule was then illuminated by a filtered xenon light (wave length: 540 nm) while rose bengal was infused intravenously. Microscopic examination revealed disintegration of the hair cells in the vestibule. Changes could be prevented by pretreatment with intravenous acetylsalicylic acid (ASA) or heparin. Twenty-four hours after the completion of photo-illumination the rats exhibited nystagmus toward the intact ear and showed rolling during the swimming test, both signs of equilibrium dysfunction. These findings were inhibited by ASA or heparin pretreatment. Our present results indicate that our method causes a photochemically induced occlusion in the rats inner ear microcirculation and may be useful for evaluating the various effects of drugs on the inner ear.

Effects of antidepressants on the intraventricular conduction and the incidence of arrhythmias induced by programmed ventricular stimulation in the dog heart after myocardial infarction

SummaryThe effects of mianserin, a tetracyclic antidepressant, and adinazolam, a new triazolobenzodiazepine which has antidepressant activity, on intraventricular conduction and the incidence of arrhythmias induced by programmed ventricular stimulation were studied in the dog heart after myocardial infarction and compared to the effects of amitriptyline, a standard tricyclic antidepressant.Amitriptyline at a dose of 1 mg/kg significantly slowed ventricular conduction in a frequency-dependent manner and at doses of 2 and 3 mg/kg significantly slowed ventricular conduction in infarcted ventricular myocardium. Amitriptyline also significantly slowed ventricular conduction in normal myocardium. Amitriptyline increased the incidence of ventricular arrhythmias induced by the programmed ventricular stimulation and prolonged the intraventricular delayed conduction resulting in re-entrant ventricular arrhythmia.On the other hand, mianserin and adinazolam at doses of 1–3 mg/kg had no significant effects on intraventricular conduction in infarcted and normal myocardium and on the incidence of arrhythmias induced by programmed ventricular stimulation. From these results, we can expect that mianserin and adinazolam may have a much lower cardiac toxicity than amitriptyline.