Impairment of cerebral cAMP-mediated signal transduction system and of spatial memory function after microsphere embolism in rats.

The transcription factor cAMP-responsive element binding protein (CREB) has been implicated in synaptic plasticity and memory. The purpose of the present study was to characterize alterations in the cAMP/protein kinase A (PKA)/CREB system after sustained cerebral ischemia. Sustained cerebral ischemia was induced by injection of 900 microspheres (48 microm in diameter) into the right (ipsilateral) hemisphere of rats. Alterations in the CREB, PKA, and cAMP levels in the cerebral cortex and hippocampus were examined up to 7 days after microsphere embolism. Immunoblotting analysis showed a decrease in the immunoreactivity of phosphorylated CREB (pCREB) in the ipsilateral hemisphere on the third day after microsphere embolism, whereas that of the total CREB was not altered. An electrophoretic gel mobility shift assay showed a decrease in the cAMP response element (CRE)-DNA binding activity of CREB in the ischemic region on the third day after the microsphere embolism. Cytosolic PKA C beta in the ipsilateral hemisphere was selectively decreased on the first day after the microsphere embolism, whereas the levels of another catalytic subunit, C alpha, and a regulatory subunit, RII alpha, were not altered. Immunoreactivity of the PKA catalytic subunit C alpha in the nucleus of the ipsilateral hemisphere was decreased on the third day after the embolism. The decreases in the pCREB, CRE-DNA binding activity, and PKA C alpha and C beta levels lasted at least up to 7 days after the operation. A decrease in the cAMP content was also seen in the ipsilateral hemisphere throughout the experiment. Furthermore, microsphere embolized rats showed prolongation of the escape latency in the water maze task determined on the seventh to ninth day after the operation. Our results suggest that sustained cerebral ischemia may impair the phosphorylation and CRE-DNA binding activity of CREB and that these effects may be one of the possible causes for learning and memory dysfunction.

Effects of terfenadine, astemizole and epinastine on electrocardiogram in conscious cynomolgus monkeys.

We examined the effects of non-sedative histamine H1 receptor antagonists on the electrocardiogram (ECG) in conscious cynomolgus monkeys. Terfenadine (3 mg kg(-1) h(-1), i.v.) and astemizole (0.3 and 1 mg kg(-1) h(-1), i.v.) caused significant time-dependent increases in the QT interval and QTc Bazett (QTc). However, normal ECG forms were found during a 60-min infusion of epinastine (3 mg kg(-1) h(-1) i.v.). A higher dose of epinastine (10 mg kg(-1) h(-1), i.v.) increased the QTc and PR interval only 5 min after the start of the infusion. The minimum plasma concentrations of terfenadine, astemizole and epinastine which caused QTc prolongation were 85, 35 and over than 3600 ng/ml, respectively. These drugs did not alter the PQ and QRS intervals and did not cause arrhythmia or atrioventricular block. Our results are consistent with the clinical observation that prolongation of QTc is caused by terfenadine and astemizole but not by epinastine. Thus, measurement of QTc in cynomolgus monkey appears to be a useful approach for evaluating the potential cardiotoxicity of histamine H1 receptor antagonists.

Synthesis and sweetness characteristics of L-aspartyl-D-alanine fenchyl esters.

Four isomers of the L-aspartyl-D-alanine fenchyl esters were prepared as potential peptide sweeteners. L-Aspartyl-D-alanine (+)-alpha-fenchyl ester and L-aspartyl-D-alanine (-)-beta-fenchyl ester showed sweetness with potencies 250 and 160 times higher than that of sucrose, respectively. In contrast, L-aspartyl-D-alanine (+)-beta-fenchyl ester and L-aspartyl-D-alanine (-)-alpha-fenchyl ester had the highest sweetness potencies at 5700 and 1100 times that of sucrose, respectively. In particular, L-aspartyl-D-alanine (-)-alpha-fenchyl ester had an excellent sweetness quality; but L-aspartyl-D-alanine (+)-beta-fenchyl ester did not have an excellent quality of sweetness because it displayed an aftertaste caused by the strong sweetness.

Comparative studies on the fate of 14C-dipyridamole (RA8) after single and multiple administration in rats.

1) Absorption, distribution and excretion of 14C-Dipyridamole (RA 8) wee studied comparatively after the administration of single intravenous (5 mg/kg), single oral (10 mg/kg) and multiple oral (10 mg/kg, once a day for one week) dosages in rats. 2) The blood level of radioactivity after oral administration showed a monophasic slow elimination with a half-life (t1/2) of about 10 hrs. The apparent absorption rate was not so high (t1/2 : 0.72 hr), therefore, it took about 3 hrs to reach the maximum level. 3) When the drug was applied intravenously, radioactivity in blood decreased tri-phasically, but the terminal phase, which appeared 12 hrs after the application, showed the same elimination rate as that of p. o. application, i.e., t1/2 : about 10 hrs. 4) The observed values of radioactivity during and after the multiple dosing regimen fitted well on the simulation curve derived from the results of a single administration. 5) Radioactivity was mainly distributed in the alimentary canal, liver and kidney, which were isolated after single or multiple oral administration. No radioactivity was found in CNS. Whole body autoradiogram obtained after intravenous or oral administration also supported the results of distribution as above. 6) No considerable accumulation of radioactivity was recognized in any tissues after the multiple dose in the rat.

Simultaneous determination of bunitrolol and its metabolite in biological fluids, plasma and urine.

A high-performance liquid chromatographic method for the simultaneous determination of bunitrolol (Koe 1366) and its metabolite, p-hydroxybunitrolol (Koe 1801) has been developed. Using the method, the sensitive and selective determination of Koe 1366 and Koe 1801 can be performed with a simple extraction with diethyl ether and spectrofluorometric detection. The detection limits of Koe 1366 and Koe 1801 in plasma are both less than 2 ng using 1-ml samples. This method was applied to human and rabbit plasma samples collected after oral administration of bunitrolol tablets (20 mg). The results show the species difference in the metabolism of bunitrolol.

Melatonin in serum and the pineal of spontaneously hypertensive rats.

Involvement of melatonin in the blood pressure regulation as an endogenous central hypotensive factor has been suggested in rats and in man. We studied the relationship between melatonin and the development of hypertension in 5- and 15-week-old spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats, by measuring serum and the pineal concentrations with a sensitive and specific radioimmunoassay coupled with a novel extraction method. Serum melatonin concentration at midnight in young SHR rats was significantly higher than that in age-matched WKY rats (P less than 0.01), whereas it was decreased in the adult SHR (P less than 0.01). No such differences were observed at noon. Pineal content of melatonin at midnight in 5-week-old SHR rats was lower than in age-matched WKY rats (P less than 0.01). These data demonstrate that melatonin in the nocturnal serum of SHR rats is elevated at prehypertensive stage while it is decreased after the development of hypertension. The role of melatonin in the hypertensive process in SHR rats requires further study.

Simultaneous determination of the enantiomers of pimobendan and its main metabolite in rat plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the simultaneous analysis of the enantiomers of pimobendan and its main metabolite, with an n-hexane-ethanol-acetic acid solvent system, has been developed. After solid-phase extraction from plasma, the enantiomers were separated from each other using a Sumichiral OA-4400 column, which is commercially available and contains a chiral stationary phase composed of (S)-proline and (S)-1-(alpha-naphthyl)ethylamine coated on silica. The enantiomers were detected with a fluorescence detector (excitation at 330 nm, emission at 415 nm). The intra- and inter-day precision studies showed good reproducibilities: the coefficients of variation were less than 10.3% for pimobendan enantiomers and 13.0% for metabolite enantiomers. The calibration curves were linear (r2 > 0.996) in the concentration range 1.25-200 ng/ml. The minimum measurable level was 125 pg per 100 microliters of plasma. The method was used in a preliminary pharmacokinetic study in three male rats after intravenous administration of racemic pimobendan (2 mg/kg).

Effects of fominoben-HCl on rat liver.

The effect of Fominoben-HCl (Noleptan) was studied in male rats given 200 mg/kg/day over one, two or four weeks. In all three experiments a slight enlargement of the liver was observed. After one-week treatment, activity of microsomal mixed-function oxidase was stimulated without an increase in cytochrome P-450 content. After 2 or 4 weeks of treatment the activity of microsomal mixed-function oxidase expressed as the activity per microsomal protein remained unchanged. The fominoben-HCl-caused biochemical changes and the slight increase in organ weight of the liver were completely reversible within 2 weeks after cessation of treatment.

Characterization of microsphere embolism-induced impairment of learning and memory function and the cholinergic system.

The impairments of learning and memory function and of the cholinergic system were examined in rats with microsphere embolism. Microsphere embolism was induced by injection of 900 microspheres with a diameter of 48 microm into the right internal carotid artery. The retention latency of a passive avoidance test was shortened and the escape latency of a water maze test was prolonged, when the animals were tested on the 5th to 10th day after the embolism, suggesting learning and memory dysfunction. Cholinergic parameters of the striatum and hippocampus, such as acetylcholine (ACh) content (67 and 60% decrease, respectively), choline acetyltransferase (ChAT) activity (45 and 56% decrease, respectively), and Bmax of muscarinic acetylcholine M1-receptor (43 and 37% decrease, respectively), were reduced on the 11th day after the embolism, suggesting attenuation of ACh synthesis and a decrease in the number of muscarinic acetylcholine M1-receptors mainly in the striatum and hippocampus. Areas not stained with triphenyltetrazolium chloride, an indication of infarction, were detected mainly in the striatum and hippocampus and partly in the frontal cortex on the 11th day after the embolism. The results suggest that an animal with microsphere embolism may be a good ischemic model with relatively sustained impairments of learning and memory function and of the striatal and hippocampal cholinergic system.

Identification of human cytochrome P450 isoforms involved in the metabolism of brotizolam.

1. To identify the cytochrome P450 (CYP) isoenzyme(s) responsible for the major metabolic pathways of brotizolam in man, we examined the metabolism of brotizolam using human liver microsomes and microsomes expressing individual human CYP isoenzymes (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4). 2. Brotizolam was metabolized to alpha-OH- and 6-OH-brotizolam by human liver microsomes (n = 3). Vmax for alpha- and 6-hydroxylation of brotizolam were 1470 +/- 259 and 8983 +/- 7740 pmol/min/mg protein respectively, and the corresponding Km were 49 +/- 9.3 and 595 +/- 580 microM respectively. 3. Among CYP inhibitors examined (furafylline, sulphaphenazole, quinidine, ketoconazole and cimetidine), ketoconazole showed the most potent inhibitory effect on brotizolam metabolism by human liver microsomes. Ki of ketoconazole for alpha- and 6-hydroxylation were 0.05 and 0.07 microM respectively. 4. When incubated with microsomes expressing individual human CYP isoenzymes (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4), brotizolam was metabolized only by CYP3A4. 5. Brotizolam metabolism in human liver microsomes was almost completely inhibited by anti-CYP3A4 antiserum. 6. These results suggest that CYP3A4 is predominantly responsible for both alpha- and 6-hydroxylation of brotizolam in human liver microsomes.

Hypotensive effect of bunitrolol at low plasma concentrations in conscious, unrestrained spontaneously hypertensive rats.

Effects of bunitrolol on mean arterial pressure (MAP) and heart rate (HR) were studied in conscious, unrestrained spontaneously hypertensive (SHR) rats at rest and during handling stress. Propranolol was employed as a reference drug. Plasma drug concentrations were determined to related with the cardiovascular effects of the drugs. Bunitrolol produced a tachycardia for the first 2 hr and a significant reduction in resting MAP at 3 and 4 hr after the oral dose (5 mg/kg) when plasma bunitrolol concentration was less than 10 ng/ml, indicating the difference between cardiac and vascular beta adrenoceptors in sensitivity to intrinsic sympathomimetic action or direct vasodilator action. Propranolol (5 mg/kg) produced no discernible effects on resting MAP and HR. Stress-induced tachycardia was significantly inhibited by both drugs throughout the experiment, while significant inhibition of hypertensive response was observed only at 4 hr after the treatment. Both bunitrolol and propranolol were rapidly absorbed from the gastrointestinal tract. Plasma half-life of these drugs were almost the same values of around 2 hr. These results indicate that dose size, plasma concentrations, and procedures and the timing of blood pressure measurement are the important factors to be considered when the antihypertensive effect of beta-blockers is studied in SHR rats.

Absorption, distribution and excretion of 14C-pirenzepine in rats. Accumulation characteristics after multiple dose regimen.

Absorption, distribution and excretion of 14C-labelled 5,11-dihydro-11-[(4-methyl-1-piperazinyl) acetyl]-6H-pyrido[2,3-b][1,4]-benzodiazepin-6-one-dihydrochloride (pirenzepine, LS-519 Cl2) were studied in SD-JCL rats. After a single i.v. injection of the drug (2 mg/kg), the concentration of radioactivity in the blood decreased bi-phasically with half-lives of 1 and 8 h. After s.c. administration of the same dose, the drug was very rapidly absorbed and it was shown that the concentrations and the elimination pattern were almost identical to that observed following i.v. injection. After oral administration of 20 mg/kg, absorption was relatively slow, taking 3 h to reach Cmax. The concentrations of radioactivity distributed in most tissues reached their maximum 3 h after a single oral administration, well comparable with that in the blood. In the study with multiple administration (5mg/kg once a day up to 14 days), 24 h after withdrawal of 7-day administration, the concentration in the liver, kidneys and testes were two times those at a 1-day administration. This ratio, however, did not increase furthermore over a 10- to 14- day administration period. All tissue levels gradually decreased with time after the final administration. Thus, no specific or considerable accumulation was demonstrated. The drug found excreted into urine was about 46% and into feces about 53% of the administered dose 96 h after i.v. injection. After single oral administration, urinary excretion was found extremely low, i.e., 8%, while 91% was excreted via feces.