Satoru Nagashima

Photochemically induced thrombosis model in rat femoral artery and evaluation of effects of heparin and tissue-type plasminogen activator with use of this model.

We report a new and reproducible model of thrombosis in the rat femoral artery. The thrombosis is initiated by endothelial injury subsequent to photochemical reaction between systemically injected rose bengal (10 mg/kg, i.v.) and transillumination of filtered xenon lamp (wave length: 540 nm) from the outside of the vessel. The blood flow of the femoral artery, which was monitored by a pulsed doppler flow meter, was fully stopped in 348.68 +/- 36.18 sec (n = 12) after i.v. injection of rose bengal under irradiation with green light. The formation of massive thrombosis was readily evident by visual inspection. The processes of primary endothelial injury and the subsequent formation of thrombosis during this manipulation were observed by light microscopy and analysed by the scanning and transmission electron microscopy. Pretreatment with heparin (30, 100 or 300 units/kg, i.v.) 10 min before rose bengal injection dose-dependently prolonged the time required to interrupt the blood flow. The thrombolytic activity of a tissue-type plasminogen activator (tPA) was also investigated. After the establishment of stable thrombotic occlusion of the femoral artery, infusion of tPA was started from the contralateral femoral vein for 30 min at the rate of 30 or 100 micrograms/kg/min. The occluded artery was reperfused in 2 out of 10 rats and in 9 out of 12 at the lower and higher rates of tPA infusion, respectively. That heparin could prevent the arterial occlusion and that tPA could reperfuse the occluded artery are observations consistent with the histopathological ones that the primary lesion of endothelium injured photochemically activates the platelet aggregation to form platelet-rich thrombus with extensions of erythrocyte-rich lesions. This model is expected to be a useful tool for evaluating the antithrombotic and thrombolytic agents.

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.

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.

Comparative antiplatelet effects of aspirin, vapiprost and GR144053, a GPIIb/IIIa antagonist, with a special reference to the role of platelet microaggregates

Microthrombi produced have a potential to form larger thrombi, leading to vascular occlusions. Recently, a new device to easily detect microaggregates using laser‐light scattering (LS) has been developed. We adopted this device to comparatively evaluate the inhibitory effects of aspirin (1, 3 or 10 mg kg−1), vapiprost (0.3, 1 or 3 mg kg−1) or GR144053 (0.1, 0.3 or 1 mg kg−1) on ex vivo aggregation of hamster platelets in relation to their in vivo antithrombotic effects. A transluminal thrombus was produced in the hamster femoral artery by the photochemical reaction. Each compound was injected i.v. as a bolus 10 min prior to the reaction, showing a dose‐dependent antithrombotic effect, i.e. they prolonged the time before the artery occluded. At that time cyclic flow reductions occurred more marked when aspirin or vapiprost was given. At the end of experiments, blood was collected to evaluate the platelet aggregation using both the new LS device and the conventional optical density (OD) method. Many more small aggregates were still formed when the highest dose of aspirin or vapiprost was used as compared with that of GR144053, although suppression of the platelet aggregation using the OD method, prolongation of the occlusion time and the bleeding time were quite similar. In conclusion, a GPIIb/IIIa antagonist markedly suppressed the microthrombi and reduced the cyclic flow reduction. This further indicates the importance of small aggregates as triggers of thrombosis and shows that prevention of their formation may result in improved vascular patency after thrombotic insult.

Antiarrhythmic effects of a benzothiazine derivative (SD-3211) and its stereoisomer (SA3212) in anaesthetized rats and isolated perfused rat hearts compared with bepridil

SummaryThe antiarrhythmic effects of a new calcium channel blocking agent (SD-3211) and its stereoisomer with additional sodium channel blocking activity (SA3212), were compared with those of a known antiarrhythmic drug (bepridil), using the left coronary artery ligation- and reperfusion-associated arrhythmia models both in isolated rat hearts and in anaesthetized rats.Isolated and perfused rat hearts were subjected to regional ischaemia for 15 min and subsequent reperfusion for 5 min. SD-3211 and SA3212 showed dose-dependently prolongations of the time interval between coronary ligation and first appearance of ventricular premature beats, reductions in the number of total ventricular premature beats during the ligation period and reductions in the incidence of reperfusion-induced ventricular fibrillation. The values of the negative logarithm of IC50 (mol/l) of SD-3211, SA3212 and bepridil were 7.97, 7.41 and 6.64 for the reduction of ventricular premature beats during ligation and 6.43, 7.49 and 6.17 for the reduction of ventricular fibrillation during reperfusion, respectively. In a separate study on force of concentration and coronary flow in perfused heart paced at 340–360 beats/min SD-3211 caused a significant negative inotropic effect between 10−7 and 10−6 mol/l. SA3212 at the concentration of < 10−6 mol/l did not result in any significant change in force of contraction. The coronary flow was increased dose-dependently by SA3212, while it was first increased and then reduced in the presence of higher concentration of SD-3211 (> 10−7 mol/l). Hearts of aneasthetized rats were also subjected to regional ischaemia for 7 min and subsequent reperfusion. SD-3211, even at the lowest dose tested (25 mg/kg), had a marked protective effect against the ligation-associated arrhythmias. The highest dosage of SA3212 tested (100 mg/kg) also reduced them. SA3212, even at the lowest dosage (25 mg/kg), resulted in a significant reduction of the incidence of reperfusion-induced ventricular fibrillation, while only the highest dosage of SD-3211 (100 mg/kg) reduced it. As for the protective effect against ligation-associated ventricular premature beats SD-3211 is about seven times as potent as bepridil, and for the reduction in the incidence of reperfusion-induced ventricular fibrillation SA3212 is about fourteen times as potent as bepridil. Significant falls in systolic blood pressure and heart rate were observed at the higher doses of SD-3211 (50 and 100 mg/kg).Thus, SD-3211 affords substantial protection against ischaemia-induced ventricular antiarrhythmias partly through the negative inotropic and chronotropic effects, and reduction of afterload. The antiarrhythmic action of SA3212 against reperfusion-induced ventricular fibrillation may be partly explained by depression of automaticity together with a reduction of the inward sodium current.

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.

Effects of DPI 201-106, a novel cardiotonic agent, on hemodynamics, cardiac electrophysiology and arrhythmias induced by programmed ventricular stimulation in dogs with subacute myocardial infarction: A comparative study with dobutamine

SummaryDPI 201-106 (DPI), a novel and potent cardiotonic agent, exhibits its effects by prolonging the open state of Na+ channels, resulting in an increase in action potential duration, and thus, is supposed to share the class III antiarrhythmic activity. The effects of DPI on the hemodynamics, intraventricular conduction and refractoriness of heart, and the incidence of arrhythmias induced by programmed electrical ventricular stimulation (PES) were compared with (±)-dobutamine. Dogs which survived for 5 to 7 days after the induction of myocardial infarction were used as the model. The presence of sub-acute myocardial infarction caused by occluding the left anterior descending coronary artery elicited a mild left ventricular dysfunction represented by a significant decrease in peak LV dp/dt by about 20%.Both i.v. bolus injection of DPI (1, 3 and 5 mg/kg) and i. v. continuous infusion of dobutamine (3, 5 and 10 μg/kg/min), which were administered in a cumulative manner, dose-dependently improved the hemodynamic parameters. At the higher doses of both DPI (3 and 5 mg/kg) and dobutamine (5 and 10 μg/kg/min) the control values were reached or even exceeded. DPI dose-dependently increased the effective refractory period (ERP) of both non-infarcted and infarcted ventricular myocardia to a similar degree, but the conduction time showed a frequency-dependent increase in the infarcted myocardium to a greater degree than in the non-infarcted myocardium after DPI. In contrast, dobutamine decreased the ERP in both non-infarcted and infarcted myocardia, and slightly increased the difference of refractoriness between the non-infarcted and infarcted zones with no effect on the intraventricular conduction. In the PES study, DPI (3 and 5 mg/kg) produced a significant decrease in the incidence of ventricular tachycardia, whereas dobutamine (5 and 10 μg/kg/min) tended to worsen the arrhythmias. These findings suggest that cardiotonic agents with a class III antiarrhythmic property such as DPI may be potentially useful for the management of heart failure accompanied by ischemic heart disease.

Pharmacokinetic and Pharmacodynamic Properties of a New Thromboxane Receptor Antagonist (Z‐335) after Single and Multiple Oral Administrations to Healthy Volunteers

The pharmacokinetics and pharmacodynamics of a new oral thromboxane (TX) A2 receptor antagonist, Z‐335, were studied in healthy male volunteers following single doses (0.5–40 mg, PO) in a dose‐escalating manner and multiple doses (40 mg, PO, once daily for 7 consecutive days) with a single‐blind, placebo‐controlled design. Serial blood and urine samples were analyzed for Z‐335 and its metabolites to obtain key pharmacokinetic parameters. In the single‐dose (10, 20, and 40 mg) study, the maximum plasma concentration (Cmax) and area under the plasma concentration versus time curve (AUC) increased in proportion to the dose when administered after fasting, while the mean elimination half‐life (t1/2β) was essentially unchanged (7.79‐7.93 h). Recovery of the unchanged and taurine‐conjugated drugs in the urine within 24 hours was 6.5% to 8.4% and 11.9% to 14.2%, respectively. These parameters essentially remained unchanged when the effect of meal intake was evaluated at the dose of 20 mg with a crossover design. Ex vivo platelet aggregation in the plasma by a TXA2 analogue, U46619, was completely inhibited within 2 hours after all doses, and complete inhibition was maintained for 12 to 14 hours, depending on the dose. The aggregation induced by collagen was also inhibited to a lesser extent, whereas that by adenosine diphosphate was hardly influenced. In themultiple‐dose study, Cmax and AUC0–24 were increased by 34% after the last dose compared with the first dose. Z‐335 afforded extensive inhibition of platelet aggregation by U46619 throughout the administration period, which returned, however, almost to the control level 48 hours after the last dose. The agent was well tolerated without any abnormalities in subjective and objective symptoms, blood biochemistry, hematology, and urinalysis definitely attributable to the agent, except for the changes expected from its TXA2 receptor‐antagonizing actions. Z‐335 was concluded to be safe and to provide long‐lasting blockade of TXA2 receptors on the basis of a once‐daily regimen, promoting further clinical evaluation.

Pharmacokinetic and Pharmacodynamic Profiles of Vapiprost, a Selective, Long‐Lasting Thromboxane Receptor Antagonist, After Single and Multiple Oral Administration to Healthy Volunteers

A selective thromboxane A2 (TXA2) receptor blocking agent, vapiprost, was orally administered to healthy male Japanese volunteers to investigate the pharmacokinetic and pharmacodynamic properties. The time‐profile of vapiprost concentration in plasma was determined and the effects of the drug on platelet aggregation in platelet‐rich plasma (PRP) induced by a stable TXA2 receptor agonist U‐46619, adenosine diphosphate (ADP) and collagen, and platelet aggregation in whole blood induced by U‐46619 ex vivo were simultaneously examined and compared. In the single‐dose study (5, 10, and 20 mg/man) the plasma concentrations of the drug were fitted well to a one‐compartment open model with a first‐order absorption. The area under plasma concentration‐time curve (AUC) and maximum plasma concentration (Cmax) showed dose‐related increases, whereas the mean elimination half‐lives (t1/2) remained approximately constant within the range of 0.99–1.1 hour. The drug was hardly recovered unchanged in urine. The platelet aggregation in PRP induced by collagen or U‐46619 and the secondary aggregation by ADP were inhibited; that induced by U‐46619 was the most specifically and completely inhibited at 2 hours after administration of any dose. The duration for maintaining the significant inhibition tended to depend on the dose and ranged from 24 to 36 hours after administration, which was much longer than expected from the plasma concentration of drug. The time‐profile of inhibiting whole blood platelet aggregation that was induced by U‐46619 was almost parallel to that of platelet aggregation in PRP by the same aggregant. The bleeding time was slightly prolonged 2 and 8 hours after administrations of 10 and 20 mg, respectively. In the multiple‐dose study (20 mg/man twice daily for 5 days; 9 doses in total) plasma concentration of the drug after each administration showed a good conformity with the simulation curve worked out using the pharmacokinetic parameters that were obtained at the time of the initial dose. This indicated that repeated administrations did not result in accumulation. Throughout the administration period, almost the same extent of inhibiting platelet aggregation by each aggregant was maintained. After the final administration almost complete inhibition of platelet aggregations both in PRP and whole blood by U‐46619 was maintained till 48 hours and a significant inhibition lasted over 72 hours, which also surpassed the duration of maintaining plasma concentration. The bleeding time showed no significant prolongation. No abnormality attributable to the test drug was found in the routine laboratory tests, subjective and objective findings, vital signs including blood pressure, pulse rate, and body temperature, and ECG except that one of six subjects in the multiple‐dose study showed a slight elevation of liver function test parameters. Drug administration was discontinued in this subject after the eighth dose. These parameters returned to the normal values within 1 week after the discontinuation. In conclusion, oral administration of vapiprost was well‐tolerated with long‐lasting inhibition of platelet aggregation both in PRP and whole blood.

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.