We synthesized various 4-(3-chloro-4-methoxybenzyl)aminophthalazines substituted at the 1- and 6-positions and evaluated their inhibitory activity toward phosphodiesterase 5 (PDE5) and their ...vasorelaxant activity in isolated porcine coronary arteries precontracted with prostaglandin F2α (10-5 M). The preferred substituents at the 1-position of the phthalazine were 4-hydroxypiperidino, 4-hydroxymethylpiperidino, 4-(2-hydroxyethyl)piperidino, and 4-oxopiperidino. Among these compounds, 4-(3-chloro-4-methoxybenzyl)amino-1-(4-hydroxy)piperidino-6-phthalazinecarbonitrile monohydrochloride (13) exhibited potent PDE5 inhibitory activity (IC50 = 0.56 nM) with >1700-fold high selectivity over other PDE isozymes (PDE1−4). Compound 13 exhibited the most potent vasorelaxant action (EC50 = 13 nM) in this series of compounds. Compound 13 reduced mean pulmonary arterial pressure by 29.9 ± 3.1% when administered intravenously at 30 μg/kg to the chronically hypoxic rats and had an apparent oral bioavailability of about 19.5% in rats and was selected for further biological evaluation.
1. The pharmacokinetic s and disposition of E4177, an angiotensin II (Ang II) type 1 receptor antagonist, were studied in the beagle dog and cynomologus monkey following intravenous (i.v.) and oral ...(p.o.) administration. The relationship between the plasma concentrations of E4177 and Ang II receptor blockade were investigated in both species.
2. Single 0.3 mg kg i.v. doses of E4177 in dog and monkey were eliminated rapidly. The elimination half-lives were 1.9 and 2.0 h, and the systematic plasma clearance values were 9.1 and 12.9 ml/min/kg respectively.
3. The oral bioavailabilityof single doses of 0.3-3 mg/kg of E4177 was > 60% in both dog and monkey. The absorption by both species was rapid, with peak plasma levels observed within 1 h, and the areas under the concentration versus time curve to infinity were proportional to the dose.
4. The apparent volumes of distributionat the steady-state were 1.0 and 1.2 l/kg in dog and monkey respectively. Tissue penetration was probably limited by the relatively high binding to plasma proteins (approximately 92.0 and 98.6% in the dog and monkey respectively).
5. Faecal excretion was the major eliminationpathway for radioactivity(approximately 90% of the dose) in both species after 1 mg/kg p.o. administration of 14C-E4177. Unchanged E4177 was the major radioactive component in the urine and faeces (0-24 h) of both species, accounting for approximately 85% of dose. In monkey, a minor metabolitein excreta and plasma was identified as the phase I metabolite M1, which is produced from E4177 by methyl- hydroxylation. M1 was not detected in dog. 6. The unbound plasma concentration versus blockade of the exogenous Ang IIinduced vasopressor response was also determined after an i.v. administrationof E4177 to dog and monkey. The relationship between the unbound E4177 concentration and the effect was highly significant in both species. The IC50 of the dog and monkey were not significantly different: 2.6 and 2.7 ng/ml respectively.
The expression of cytochrome P450 3A4 (CYP3A4) in the foveolar epithelium of the human stomach with intestinal metaplasia was studied using immunohistochemistry, western blotting and reverse ...transcription polymerase chain reaction (RT‐PCR). CYP3A4 was immunohistochemically detected in the foveolar epithelium with intestinal metaplasia, but was not detected in foveolar epithelium without intestinal metaplasia, in the pyloric gland or in the fundic gland of the stomach. Western blotting and RT‐PCR demonstrated that CYP3A4 protein and mRNA were expressed in the liver and pyloric gland mucosa with intestinal metaplasia, but not in the fundic gland mucosa without intestinal metaplasia. Possible roles of CYP expression in the gastric mucosa with intestinal metaplasia in human stomach carcinogenesis are briefly discussed.
We investigated the distribution of a novel angiotensin II type 1 (AT1) receptor antagonist, E4177 (4'-2-cyclopropyl-7-methyl-3H-imidazo5, 4-bpyridine-3-ylmethyl-2-biphenylcarboxylic acid), in rat ...adrenal glomerulosa. In a binding assay of adrenal capsular tissue (mainly glomerulosa), E4177 exhibited a maximum displacement of approximately 80% of total 125I-labeled angiotensin II (125I-Sal1, Ile8 Ang II) binding, and its IC50 value was 6.9±0.5 nM. This IC50 value indicated a slightly higher in vitro potency than that of losartan (21.0±0.6 nM). Also, in a receptor autoradiographic study, E4177 (10000 nM) displaced approximately 80% of radiolabeled 125I-Sal1, Ile8 Ang II in rat adrenal glomerulosa and caused only slight displacement in rat adrenal medulla. Further, light and electron microscopic autoradiography of adrenal glomerulosa for 15 min after the intravenous administration of 1 mg/kg 14CE4177, indicated the localization of 14C, possibly in the adrenal zona glomerulosa cell plasma membrane. It was strongly suggested that E4177 is a potent and selective antagonist of the AT1 receptor, and that it specifically binds to AT1 receptors in the adrenal zona glomerulosa.
Changes in the mRNA levels for aldosterone synthase cytochrome P450 (P450aldo or CYP11B2) in rat adrenal glands were studied in response to angiotensin II type 1 (AT1) and type 2 (AT2) receptor ...antagonists. Since 11 beta hydroxylase P450 (P45011 beta or CYP11B1), which shows high homology (88.5%) with P450aldo in their nucleotide sequences of the amino acid coding regions, is also expressed in the adrenal gland, RT-PCR was performed with specific primers for each P450. Upon sodium restriction (5 mmol Na+/kg of diet) of the rats for 14 days, the amount of the P450aldo mRNA in the adrenal glands increased 8.5 fold above from the rats fed on a normal diet (225 mmol Na+/kg of diet), whereas no significant change of the P45011 beta mRNA was observed after the dietary sodium restriction. As shown by an immunoblot analysis, the adrenal capsule portions (mainly zona glomerulosa) of the rats kept on the low Na diet for 14 days expressed significantly higher level of P450aldo than those from the rats fed the normal diet. In concert with the alteration, plasma aldosterone concentration increased. However, when a specific AT1 antagonist E4177 was given to the rats kept on a low Na diet, the amount and activity of P450aldo as well as the plasma aldosterone concentration was suppressed. On the other hand, the increase of P450aldo induced by the low Na diet was not affected by an AT2-specific antagonist, PD123177.
Changes in the mRNA levels for aldosterone synthase cytochrome P450 (P450aldo or CYP11B2) in rat adrenal glands were studied in response to angiotensin II type 1 (AT1) and type 2 (AT2) receptor ...antagonists. Since 11β hydroxylase P450 (P45011β or CYP11B1), which shows high homology (88.5%) with P450aldo in their nucleotide sequences of the amino acid coding regions, is also expressed in the adrenal gland, RT-PCR was performed with specific primers for each P450. Upon sodium restriction (5 mmol Na+ /kg of diet) of the rats for 14 days, the amount of the P450aldo mRNA in the adrenal glands increased 8.5 fold above from the rats fed on a normal diet (225 mmol Na+ /kg of diet), whereas no significant change of the P45011β mRNA was observed after the dietary sodium restriction. As shown by an immunoblot analysis, the adrenal capsule portions (mainly zona glomerulosa) of the rats kept on the low Na diet for 14 days expressed significantly higher level of P450aldo than those from the rats fed the normal diet. In concert with the alteration, plasma aldosterone concentration increased. However, when a specific AT1 antagonist E4177 was given to the rats kept on a low Na diet, the amount and activity of P450aldo as well as the plasma aldosterone concentration was suppressed. On the other hand, the increase of P450aldo induced by the low Na diet was not affected by an AT2-specific antagonist, PD123177.
A stereoselective HPLC method has been developed for the determination of E2011 (compound I) and one of its metabolites and diastereoisomers, ER-20593 (compound II), in plasma. The two substances and ...the internal standard were extracted from plasma, followed by two purification steps. In the first step, a minicolumn, Bond Elut C
1
8, was used and in the second step, another minicolumn, Bond Elut Si, was used for purification of the compounds. After the purification, the compounds were analyzed by HPLC with two Chiralpak AD columns. In this procedure, compounds I and II were stable and there was no chiral inversion. The within-day and the between-day assays were performed in rat plasma, where compounds I and II existed simultaneously. The within-day and the between-day precisions of compound I were 2.0∼10.1% and 1.3∼7.1%, and the within-day and the between-day accuracies were −8.2∼+3.0% and −6.6∼+4.0% in the concentration range 0.003–10 μg
ml
−1. The within-day and the between-day precisions of compound II were 1.7∼16.9% and 0.9∼4.5% and the within-day and the between-day accuracies were −9.0∼+2.4% and −5.6∼+3.8% in the concentration range of 0.005-0.5 μg
ml
−1. QC samples for compound I and II were stable for at least 3 months. The method was applied to measure the levels of compound I and II in the rat plasma after oral administration of compound I.
After oral administration of super(14)C-labelled (5R)-3-2-((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl-5-metho x ymethyl-2-oxazolidinone (E2011) at a dose of 1 mg/kg, the blood level of ...radioactivity reached a maximum concentration (C sub(max)) of 0 super(.)545 mu g eq./ml after 0 super(.)25 h in the rat and of 0 super(.)900 mu g eq. /ml after 0 super(.)5 h in the dog. In dog plasma, C sub(max) for radioactivity and unchanged E2011 were 0 super(.)862 mu g eq./ml and 0 super(.)650 mu g/ml respectively with corresponding T sub(max) (time at C sub(max)) of 0 super(.)75 and 0 super(.)25 h. The unchanged drug in dog plasma was below the detection limit (5 ng/ml plasma) after 24 h. The tissue levels of radioactivity were measured at 0 super(.)25 (T sub(max)), 6, 24, and 168 h after administration to the rat and at 0 super(.)5 (T sub(max)), 24, and 168 h in the dog. The radioactivity was distributed in all tissues examined at T sub(max) in the rat and dog. The radioactivity levels of the cerebral cortex in the rat and dog were 26 and 36% of the plasma level at T sub(max). The radioactivity in tissues decreased at almost the same rate as that in plasma. Plasma protein binding of the unchanged drug in the rat in vitro were about 70% in the range of 0 super(.)1-10 mu g/ml, and those in the dog were about 45% in the same concentration range. Cumulative excretion of radioactivity in the rat was 74 super(.)5% in urine and 22 super(.)5% in faeces after 7 days. In the dog, 55 super(.)5 and 36 super(.)5% of the radioactivity administered were excreted in urine and faeces respectively after 7 days. The biliary excretion of radioactivity in the cannulated rat was 23 super(.)0% within 48 h. In tlc analysis of plasma and tissues of the rat and dog, the radioactivity for the unchanged drug was much higher than metabolites. In tlc analysis of urine, the same metabolites were detected in the rat and dog, and the radioactivity of a metabolite, IM1, was the highest in the both animals. Eight metabolites were detected in the plasma, tissues and excreta of the rat, and four metabolites in the dog. In conclusion, the absorption, distribution, metabolism and excretion of super(14)C-labelled E2011 in the rat and dog have been established, and only minor differences were observed between these species.
Porcine heart tissue-type plasminogen activator (t-PA) was reconstituted onto large multilamellar liposomes with various lipid compositions and the kinetics of plasminogen activation by free or the ...reconstituted t-PA were studied. Negatively charged lipids, sulfatide and phosphatidylserine (PS), lowered the Km values of t-PA for plasminogen activation (sulfatide, 20-fold; PS, 6-fold), whereas neutral lipid phosphatidylcholine raised the Km. On the other hand, these lipid environments did not affect the amidase parameters and fibrin-binding potency of t-PA. The present results suggest that t-PA could function as a cell-associated form and its plasminogen activation may be regulated by the net charge of the head group of membrane lipids.
PDF
