Jun-ichi Imagawa

Myocardial protection afforded by nicorandil and ischaemic preconditioning in a rabbit infarct model in vivo

We previously showed that preoperative nicorandil, a hybrid potassium channel opener and nitrate compound, conferred cardioprotective effects in a hypoxia/reoxygenation model of isolated human atrial muscle by using functional recovery as an end point, and that ischaemic preconditioning surprisingly abolished the protection afforded by nicorandil. In view of this paradoxic result, this study was undertaken to assess whether ischaemic preconditioning influences any protective effect of nicorandil by using infarct size as an end point. In addition, we investigated the underlying mechanisms of the protective action of nicorandil. Rabbits underwent a midline sternotomy under anaesthesia. A left coronary branch was occluded for 30 min followed by 120 min of reperfusion. Nicorandil (100 microg/kg bolus + 10 microg/kg/min) was given intravenously 30 min before coronary occlusion and continued to the time of reperfusion (early treatment) or 5 min before reperfusion and continued throughout reperfusion (late treatment). Ischaemic preconditioning was achieved by a single episode of 5-min coronary occlusion followed by 10-min reperfusion before the 30-minute occlusion in the presence or absence of nicorandil. Risk volume and infarct volume were determined by fluorescent microspheres and tetrazolium staining, respectively. Early treatment with nicorandil conferred a significant decrease in percentage of infarct size within the risk zone (24.9 +/- 2.9%) when compared with control (39.2 +/- 4.3%; p < 0.01). Late treatment with nicorandil had no effect on infarct size (43.5 +/- 3.4%). Ischaemic preconditioning also resulted in significant reduction in infarct size (13.4 +/- 4.3%; p < 0.01 vs. control). The combination of ischaemic preconditioning with nicorandil (early treatment) showed an intermediate protective efficacy between early treatment with nicorandil alone and ischaemic preconditioning alone (18.1 +/- 4.2%; p < 0.01 vs. control). Nitroglycerin (10 microg/kg bolus + 1 microg/kg/kg/min, i.v.) given before and during ischaemia tended to reduce infarct size, but the effect was not statistically significant (28.9 +/- 2.9%; p > 0.05 vs. control). Although an adenosine triphosphate (ATP)-sensitive potassium channel blocker, 5-hydroxydecanoate (5 mg/kg, i.v.) by itself had no effect on infarct size (38.8 +/- 3.6%), the protective effect of nicorandil was abolished by 5-hydroxydecanoate (37.7 +/- 5.8%; p < 0.05 vs. early treatment of nicorandil). There were no differences in area at risk or haemodynamics between groups. Our results show that nicorandil has a protective effect against myocardial infarction in our rabbit model when infused before and during ischaemia, but not during reperfusion, and the protective effect is abolished by an ATP-sensitive potassium channel blocker. Furthermore, the addition of ischaemic preconditioning does not detrimentally influence the effect of nicorandil. This suggests that nicorandil can confer an infarct-limiting effect by opening of ATP-sensitive potassium channels with or without intermittent ischaemia, as may happen in patients with unstable angina.

Further evaluation of the selectivity of a novel antihypertensive agent, SGB-1534, for peripheral α1-adrenoceptors in the spinally anestherized dog

Experiments were designed to examine some characteristics of an orally active antihypertensive agent, SGB-1534 on alpha-adrenoceptors in spinally anesthetized dogs. In the saphenous arterial bed perfused by a constant pump volume, saphenous nerve stimulation and bolus applications of norepinephrine and phenylephrine into the artery-evoked frequency- or dose-dependent increases (i.e. vasoconstriction) in perfusion pressure. SGB-1534 and prazosin infused i.v. significantly reduced the vasoconstriction in response to saphenous nerve stimulation and the two agonists. In the saphenous arterial bed, alpha 1-adrenoceptor antagonist potency of SGB-1534 on a weight basis was approximately 30 times greater than that of prazosin. Unlike SGB-1534 and prazosin, yohimbine failed to inhibit the vasoconstriction induced by phenylephrine, but instead potentiated the vasoconstrictor response to saphenous nerve stimulation. The equieffective doses of methoxamine and B-HT 920 given i.v. produced sustained pressor responses. SGB-1534 and prazosin applied i.v. in a cumulative way reduced dose dependently the pressor response to methoxamine but not that to B-HT 920. When the doses that blunted the sustained pressor response to methoxamine by 50% were compared, the alpha 1-adrenoceptor antagonistic activity of SGB-1534 was nine times greater than that of prazosin.

Spasmolytic Action of Nicorandil in Canine Conductive Coronary Arteries In Vivo Is Not Modified by Glibenclamide

The spasmolytic effects of nicorandil, cromakalim, and nitroglycerin on coronary arteries were investigated by angiographic technique in anesthetized dogs. With intracoronary arterial (i.a.) U 46619, a thromboxane A2 mimetic, the diameter of coronary arteries decreased in a sustained manner by 36.1 +/- 1.6% from control levels (coronary spasm). With a successive i.a. injection of nicorandil (300 micrograms), cromakalim (30 micrograms), or nitroglycerin (3 micrograms), the diameter recovered control levels (102.9 +/- 3.9, 96.8 +/- 5.6, and 100.1 +/- 4.3%, respectively). In dogs treated intravenously (i.v.) with glibenclamide, a pharmacologic antagonist of K-channel openers, the spasmolytic effect of cromakalim was significantly reduced, whereas the activity of nicorandil or nitroglycerin remained unaffected. We also investigated a possible modification by glibenclamide of the increase in coronary blood flow (CBF) induced by i.a. nicorandil and cromakalim in anesthetized dogs. The dose-dependent blood flow responses to cromakalim and nicorandil were significantly attenuated by glibenclamide, whereas the response to nitroglycerin remained unaffected. These results suggest that the spasmolytic effect of nicorandil on canine conductive coronary vessels is not mediated by K-channel opening but by a nitroglycerin-like action and that the dilatation of resistive coronary vessels induced by nicorandil may be largely due to its action as a K-channel opener.

6-Substituted 2,2-bis(fluoromethyl)-benzopyran-4-carboxamide K+ channel openers.

In the course of our study to find an ideal antihypertensive potassium channel opener (KCO), N-(2-cyanoethyl)-2,2-bis(fluoromethyl)-6-pentafluoroethyl-2H-1-ben zopyran-4-carboxamide (13f, KC-515) showed a highly potent, slow and long-lasting antihypertensive effect with reduced reflex tachycardia, together with the beneficial effects of KCO such as improvement in lipid metabolism. These profiles identify KC-515 as a potential candidate. In conscious spontaneously hypertensive rats (SHR), the onset of the hypotensive effect of KC-515 (13f) was gradual and the maximum response was attained at around 6 h after dosing. The duration of action was over 18 h for 0.1 mg/kg. When administered to Zucker rats for 2 weeks with 0.03-0.3 mg/kg po range in the antihypertensive doses in hypertensive rat models, KC-515 (13f) significantly and dose-dependently reduced serum triglycerides to less than 70% of control without affecting total cholesterol.

Regional and species differences in glyburide-sensitive K+ channels in airway smooth muscles as estimated from actions of KC 128 and levcromakalim

1 The purpose of the present experiments was to elucidate the differences in actions of two K+ channel openers, KC 128 and levcromakalim, on the carbachol‐induced contraction, membrane potential and 86Rb+ efflux of the dog tracheal and bronchial smooth muscles. Furthermore, we compared the effects of these agents on guinea‐pig and human airway smooth muscles. 2 In the dog tracheal and bronchial smooth muscle tissues, levcromakalim induced a concentration‐dependent relaxation of the carbachol‐induced contraction. The IC50 values were 0.35 μm (pIC50: 6.46 ± 0.10, n = 9) and 0.55 μm (pIC50: 6.26 ± 0.07, n = 5), respectively. KC 128 relaxed bronchial smooth muscles precontracted by carbachol with an IC50 value of 0.19 μm (pICso: 6.73 ± 0.10, n = 7). However, KC 128 had almost no effect on the contraction evoked by carbachol in the trachea (IC50 > 10 μm). The relaxations induced by levcromakalim and KC 128 were antagonized by glyburide (0.03‐1 μm) but not by charybdotoxin (100 nm). 3 Levcromakalim (1 μm) hyperpolarized the membrane of both dog tracheal and bronchial smooth muscle cells, whereas KC 128 (1 μm) hyperpolarized the membrane of bronchial but not of tracheal smooth muscle cells. 4 Levcromakalim (10 μm) increased 86Rb+ efflux rate from both tracheal and bronchial smooth muscle tissues but KC 128 (10 μm) increased 86Rb+ efflux rate only from bronchial and not tracheal smooth muscle tissues. Glyburide (1 μm) prevented the hyperpolarization and the 86Rb+ efflux induced by these agents at the same concentration as observed for mechanical responses. 5 Both KC 128 and levcromakalim relaxed the guinea‐pig isolated tracheal smooth muscles precontracted by carbachol (100 nm), histamine (3μm) or U46619 (10 nm). KC 128 was approximately 10 times more potent than levcromakalim for each agonist. 6 In human bronchial smooth muscles, levcromakalim but not KC 128 induced a concentration‐dependent relaxation of the carbachol‐induced contraction. 7 It is concluded that KC 128 has relaxant and hyperpolarizing effects in the dog bronchial and guinea‐pig tracheal smooth muscles, but not in the dog tracheal and human bronchial smooth muscles. On the other hand, levcromakalim acts consistently on all the above airway smooth muscle tissues. These results indicate that there are regional and species differences in distribution of K+ channels, and at least two different K+ channel opener‐ and glyburide‐sensitive K+ channels are present in the dog airway smooth muscles.

Synthesis and antihypertensive activity of KC-399, a benzopyran K+ channel opener with long duration of action and less tachycardia

Abstract Synthesis and antihypertensive activity of KC-399 (5) have been described. KC-399 (5) showed a highly potent, slow and long-lasting antihypertensive effect with reduced reflex tachycardia.

betaARK1 inhibition improves survival in a mouse model of heart failure induced by myocardial infarction.

Heart failure (HF) is characterized by abnormalities in β-adrenergic receptor (βAR) signaling, including an increase in βAR kinase 1 (βARK1) levels and activity. Gene therapy using a peptide inhibitor of βARK1 (βARKct) in infarcted rabbit hearts has improved compromised cardiac function. To determine whether βARK1 inhibition improves survival in a mouse model of HF induced by myocardial infarction (MI), we studied wild-type (WT) and transgenic (TG) mice overexpressing βARKct following MI. There was no difference in infarct size. Survival of WT mice with MI was 25% at 26 weeks. In contrast, 92% of βARKct TG mice with MI survived (P = 0.01). βARKct TG mice with MI at 8 weeks showed significantly higher fractional shortening compared with WT mice with MI (25.1 ± 2.7% versus 14.2 ± 1.0%; P < 0.05). Moreover, the biochemical βAR abnormalities in WT mice with MI were prevented in βARKct TG mice with MI. In conclusion, βARK1 inhibition results in a marked increase in survival and improved cardiac function in a mouse model of HF induced by MI.

The effect of a novel benzopyran derivative, KC 399, on the isolated guinea-pig trachealis and human bronchi.

1. In isolated guinea-pig trachealis, KC 399, BRL 38227 and salbutamol suppressed the spontaneously generated tone in a concentration-dependent manner with pD2 values of 8.89 +/- 0.09 (n = 14), 6.18 +/- 0.07 (n = 11) and 7.72 +/- 0.12 (n = 8), respectively. 2. The bronchodilator effects of KC 399 and BRL 38227 were antagonized by glibenclamide but not by charybdotoxin or apamin. The effect of salbutamol was antagonized by charybdotoxin but not by glibenclamide or apamin. 3. KC 399 and BRL 38227 failed to inhibit the tone evoked by 90 mM K+ in guinea-pig trachealis, whereas salbutamol did inhibit it, in a concentration-dependent manner. 4. These bronchodilators also relaxed the tone of isolated guinea-pig trachealis supported by histamine, carbachol, U46619 or leukotriene D4. Their order of potency was always KC 399 > salbutamol > BRL 38227. 5. KC 399 and BRL 38227 relaxed isolated human bronchi contracted with histamine or carbachol. 6. We conclude that KC 399 is a potent relaxant of isolated guinea-pig trachealis and human bronchi in vitro. The relaxant action of KC 399 could be due to the opening of glibenclamide-sensitive K+ channels.

In vivo experiments for the evaluation of α1-adrenoceptor antagonistic effects of SGB-1534 on canine urethra

The alpha 1-adrenoceptor blocking effects of SGB-1534 on the urethral smooth muscle were compared in in vivo lower urinary tract preparations of anesthetized dogs, with the effects of other alpha 1-adrenoceptor antagonists, prazosin and bunazosin. Hypogastric nerve stimulation and selective administration of phenylephrine to the urethra and bladder through the cannulated right external iliac artery (i.a.) elicited reproducible frequency- and dose-dependent increases in intra-urethral pressure. Intra-bladder pressure was increased by the nerve stimulation but not by i.a. phenylephrine. SGB-1534, prazosin or bunazosin (0.1-10 micrograms/kg i.v.) dose dependently suppressed the urethral contraction evoked by the nerve stimulation and i.a. phenylephrine but did not influence the bladder contraction elicited by nerve stimulation. The alpha 1-adrenoceptor blocking potency of SGB-1534 was approximately 2.3 and 8.1 times greater than that of prazosin and bunazosin, respectively. The results indicate that alpha 1-adrenoceptors may mediate mainly the urethral contraction induced by hypogastric nerve stimulation and i.a. phenylephrine, and that SGB-1534 was more potent alpha 1-adrenoceptor blocking activity than prazosin and bunazosin in the canine urethra.

SYNTHESIS AND VASORELAXANT ACTIVITY OF 2-FLUOROMETHYLBENZOPYRAN POTASSIUM CHANNEL OPENERS

The synthesis and vasorelaxant activity of 2-fluoromethylbenzopyran potassium channel openers are described. These (2-fluoromethyl) derivatives displayed smooth muscle relaxant activities comparable to or more potent than the corresponding 2-methyl analogues.