Naunyn-Schmiedeberg's Arch Pharmacol (1991) 344: 235 - 239

Naunyn-Schmiedeberg's

0028129891001374

Archivesof Pharmacology © Springer-Verlag1991

2-Nicotinamidoethyl acetate (SG-209) is a potassium channel opener: Structure activity relationship among nicorandil derivatives Takaharn Ishibashi, Masami Hamaguchi, and Shoichi Imai Department of Pharmacology, Niigata University School of Medicine, Asahimachi-Dori 1-757, Niigata 951, Japan Received December 31, 1990/Accepted May 13, 1991

Summary. The mechanism of the vasodilating action of 2-nicotinamidoethyl acetate (SG-209), a derivative of nicorandil, was examined in the isolated rabbit aorta. Comparison was made using 2-nicotinamidoethyl alcohol (SG-86) and 2-nicotinamidoethyl nitrate (nicorandil; SG-75) to reveal any structure-activity relationships. SG-209 and nicorandil caused concentration-dependent relaxation in preparations precontracted with phenylephrine (10 .7 mol/1), while SG-86 produced a relaxation only at very high concentrations. The pDz values (-log[ECso]) of SG-209 and nicorandil were 3.59 +_ 0.07 and 5.95 __ 0.10, respectively. The vasorelaxant activity of nicorandil was associated with significant increases in cyclic G M P content, while that of SG-209 was not. Methylene blue (10 .5 mol/1) attenuated the relaxant effect of nicorandil, but had no effect on that of SG-209. Furthermore, the relaxant effect of nicorandil was not affected by glibenclamide (10 .5 mol/1), whilst the relaxant effect of SG-209 was abolished by this compound. In the presence of methylene blue (10 .5 mol/1), however, glibenclamide (I 0 - 5 tool/l) attenuated the relaxant effect of higher concentrations of nicorandil (_> 10- s tool/l). These results indicate that the relaxant effect of SG209 is mostly if not exclusively due to the activation of potassium channels, while this action contributes to the vasodilating action o f nicorandil only at higher concentrations.

Key words: SG-209 - Nicorandil - SG-86 - Cyclic

characteristics (Nakagawa et al. 1979; Imai et al. 1983, 1987; Taira 1987). It has a nitrate moiety, essential for its pharmacological activities (Taira et al. 1979; Sakai et al 1980), and can be classified as an organic nitrate. Indeed, several investigators have provided evidence in support of the hypothesis that nicorandil causes vasodilation through activation of soluble guanylate cyclase and resultant accumulation of cyclic G M P (Holzmann 1983; Kukovetz and Holzmann 1987) in vascular smooth muscle. However, another pharmacological property of nicorandil, i.e., its potassium channel opening activity is believed to be responsible for vasodilatation of the canine mesenteric artery (Inoue et al. 1984) and the porcine and guinea-pig coronary arteries (Furukawa et al. 1981). A derivative of nicorandil, SG-209 (2-nicotinamidoethyl acetate), which does not possess a nitrate moiety, dilates tracheal smooth muscle and increases tracheal blood flow in anesthetized dogs (Maruyama et al. 1982), although its mechanism of action is unknown. Methylene blue has been accepted as a blocker of the activation of soluble guanylate cyclase by nitro-vasodilators (Martin et al. 1985), while recent studies (Cavero et al. 1989; Eltze 1989; Yamada et al. 1990) have indicated the specific inhibition of the opening of a potassium channel by glibenclamide. Using these two tools, we undertook to clarify the mechanism of the vasodilating activity of SG-209 and tried to elucidate the basic chemical structure necessary for the activation of potassium channels by pyridine-type potassium channel openers.

G M P - Isolated rabbit aorta

Methods Introduction Nicorandil (2-nicotinamidoethyl nitrate; SG-75) is a vasodilator agent (Uchida et al. 1978) with unique Send offprint requests to T. Ishibashi at the above address

Male Japanese white rabbits (2.5-4.3 kg) were treated with reserpine (2 mg/kg, i.p.) 18-24 h before sacrifice, as it is known that methylene blue produces the contraction of aortic ring preparation by releasing norepinephrine from the intramural nerves (Martin et al. 1975; Matsuoka et al. 1987). The animals were given sodium pentobarbital (25 mg/kg, i.v.) and bled. The thoracic aorta was immediately excised, placed in a chamber filled with cold KrebsHenseleit solution and dissected free of excess fat and connective

236 tissue. All animals were dealt with in a humane way in accordance with recognized guidelines on animal experimentation.

O II

Recordings of mechanical activity. The aorta was cut into rings about 3 mm wide and the endothelium was removed by a stainless steel rod. The preparations were mounted in 10 ml organ baths kept at 37°C and were equilibrated at a resting tension of 1 g for 6 0 90 min during which time the bathing solution was replaced repeatedly. Phenylephrine (10 -7 mol/1) was administered several times to obtain a maximum contraction and the removal of endothelium was confirmed by observing no relaxation with 10-6 mol/1 acetylcholine. Finally, the aortae were contracted with phenylephrine (10 -7 mol/1) and about 30 min later, the test agents were administered cumulatively. Methylene blue, glibenclamide and vehicle for glibenclamide (ethyl alcohol) were administered 30 rain after phenylephrineand another 15 rain was allowed before cumulative administration of the test compounds to obtain a steady level of tension, pA2 values were calculated according to the method of van Rossum (1963). Isometric tension changes of the ring preparations were measured by the use of strain gauges (T7-30-240, Orientec, Tokyo, Japan) and were recorded on a potentiometric recorder (Hitachi Recorder 050, Hitachi, Tokyo, Japan or Servocorder SR6211, Graphtec, Tokyo, Japan) via a strain gauge amplifier (San-ei 6M82, Tokyo, Japan).

Nicorandil

(SO - 75)

0 II C~N~O-~c/CH*

~

SG - 209 o [I

Fig. 1. Chemical structures of nicorandil, SG-209 and SG-86

20

Measurement of cyclic GMP content. The trimmed aorta was cut into rings about 15 mm wide and the endothelium was removed as mentioned above. After equilibration in Krebs-Henseleit solution, phenylephrine (10 -v mol/1) was added and after 30 rain, vasodilators were administered. Then, the preparations were frozen at a temperature of liquid nitrogen at desired times and stored at - 80° C until the time of analysis. Frozen tissue was homogenized using a Polytron PT10 (Kinematica, Luzern, Switzerland) with 1.5 ml of HC1 (0.1 N). After boiling for 3 rain, the homogenate was centrifuged for 30 min at 4000 rpm and the supernatant was used for assay. The quantitative assessment of cyclic GMP was performed by a radioimmunoassay method with a cyclic GMP assay kit (Yamasa Shoyu Co., Ltd., Choshi, Japan). Using the HC1 extract, its protein content was determined by a modified Lowry method (Peterson 1977) and its cyclic GMP content was expressed as fmol/mg protein. Solution. The ionic composition of the Krebs-Henseleit bicarbonate solution was as follows (in millimolar): NaC1, 118; KC1, 4.7; CaCI2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25; and glucose, 11.1. The solution was aerated with 95% O2 + 5% CO2 and pH was adjusted to 7.4 at 37°C. Drugs. The compounds tested were nicorandil (2-nicotinamidoethyl nitrate, SG-75) and its derivatives: SG-209, 2-nicotinamidoethyl acetate and SG-86, 2-nicotinamidoethyl alcohol (Chugai Pharmaceutical Co., Tokyo, Japan). The chemical structures of these compounds are shown in Fig. 1. These compounds as well as methylene blue (Wako Pure Chemical Industries Co., Ltd, Osaka, Japan) were dissolved in distilled water. Glibenclamide (Sigma Chemicals Co., St. Louis, MO, USA) was dissolved in 100% ethyl alcohol (2.5 mmol/1). Solutions of phenylephrine hydrochloride (Kowa Company Ltd., Nagoya, Japan) and acetylcholine chloride (Daiichi Seiyaku Co., Ltd., Tokyo, Japan) were freshly prepared before each experiment, and were diluted with distilled water before use. Statistics. The results were expressed as mean value + SEM. Statistical assessment of the significance among groups was made by oneway analysis of variance followed by Scheffe's method. Differences were considered significant at a probability value of less than 0.05.

~ 4o" ~ 6080"

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100 I

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7

6

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8 Nicorandil ( - l o g [mol/1]) 5

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2

1

Fig. 2. Effects of methylene blue and glibenclamide on the nicorandil-induced relaxation of isolated rabbit aorta. When the muscle relaxed from the steady tension just before drug administration to the resting tension before phenylephrine (10 -7 mol/1), the relaxation was taken as 100%. ©, control ( n = 6 ) ; 0 , after administration of methylene blue (10 -5 mol/1) (n = 6); A, after ethyl alcohol (final concentration of 0.4%) as a vehicle control for glibenclamide (n = 6); A, after glibenclamide (10-5 tool/l) (n = 6); [~, after administration of methylene blue (10 -5 tool/l) and glibenclamide (10-5 mol/1) (n = 7)

Results

Vasorelaxant effects of nicorandil, SG-209 and SG-86 on phenylephrine-induced contraction As s h o w n in Figs. 2 - 4 , the c u m u l a t i v e a p p l i c a t i o n o f nicorandil (10-7mol/1 1 0 - 2 t o o l / I ) a n d SG-209 (10 - 7 mol/1 - 3 x 10 - 2 mol/1) i n h i b i t e d the sustained c o n t r a c t i o n s i n d u c e d b y 1 0 - 7 mol/1 p h e n y l e p h r i n e in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r . O n the other h a n d , SG-86 did n o t cause r e l a x a t i o n u n t i l a t h r e s h o l d c o n c e n t r a t i o n o f 3 × 1 0 - 3 mol/1 was achieved. T h e r a n k o r d e r o f

237

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,~

60

80

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6

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5 4 3 SG - 209 ( - log [mol/l])

i

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Fig. 3. Effects of methylene blue and glibenclamide on the SG-209induced relaxation of the isolated rabbit aorta. Relaxation from the steady tension just before drug administration to the tension before phenylephrine (10-v tool/l) was taken as 100%. O, control (n = 6); O, after administration of methylene blue (10 -5 tool/l) (n = 5); A, after ethyl alcohol (final concentration of 0.4%) as a vehicle control for glibenclamide (n = 5); A, after glibenclamide (10 -2 mol/1) (n = 6)

0.

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80-

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i

i

i

i

i

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7

6

5

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SG - 86 (- log [tool/l])

Fig. 4. Effect of SG-86 on the rabbit aorta and the influences of methylene blue and glibenclamide thereupon. Relaxation from the steady tension just before drug administration to the tension before phenylephrine (10 .7 tool/l) was taken as 100%. O, control (n = 6); 0 , after methylene blue (10 -s mol/1) (n = 6); A, after ethyl alcohol (final concentration of 0.4%) as a vehicle control for glibenclamide (n = 6); A, after glibenclamide (10-s tool/l) (n = 6)

relaxant potency calculated from the pD2 (-log[ECso]) values obtained from the concentration-response curves was 5.95 _ 0.10 for nicorandil > 3.59 _+ 0.07 for SG-209 > 2.05 +_ 0.05 for SG-86 (n = 6 in each).

Effects of methylene blue and glibenclamide on vasorelaxant effects of nicorandil, SG-209 and SG-86 In another series of experiments, methylene blue (10 5mol/1) and/or glibenclamide (10-Stool/I) was added 30 min after administration of phenylephrine (10 .7 mol/l). All preparations responded to methylene

blue with a small increase in tone (27.2% _+ 2.3%; n = 18). However, in our preliminary studies, this increase in tone did not affect the relaxant effect ofpapaverine (data not shown). Therefore, the inhibitory effects of methylene blue on relaxations produced by the test agents were assessed by comparing the relaxations in the presence and absence of methylene blue. Glibenclamide also induced a small increase in tone (16.0% _+_ 1.7% ; n = 18), but this was not significantly different from that produced by the vehicle alone (ethyl alcohol, final concentration of 0.4%) (16.3% ± 2.8%; n = 18). The pD2 (-log[ECso]) value for nicorandil was 5.67 _+ 0.12 (n = 6) in the absence of glibenclamide (vehicle alone) and 5.71 + 0.11 (n = 6) in the presence of 10- s mol/1 glibenclamide. Thus, neither ethyl alcohol nor glibenclamide affected the relaxant effect of nicorandil. Methylene blue (10-5 tool/l) inhibited the relaxant effect of nicorandil and a parallel right-ward shift was observed giving an apparent pA2 value of 5.81 (n = 6). When both methylene blue (10 .5 tool/l) and glibenclamide (10-s mol/1) were used together, a further inhibition was observed, although only at higher concentrations of nicorandil (Fig. 2). The relaxant effect of nicorandil in the presence of methylene blue was not affected by the vehicle for glibenclamide (ethyl alcohol, 0.4%; data not shown). Neither methylene blue (10 -5 mol/1) nor the vehicle for glibenclamide (ethyl alcohol, 0.4%) affected the relaxant effect of SG-209 (Fig. 3). The pD2 value for SG-209 was 3.54 __+0.08 (n = 6) in the presence of methylene blue and 3.37 __ 0.08 (n = 6) in the presence of ethyl alcohol. In contrast, glibenclamide (10-s mol/1) inhibited the relaxant effect of SG-209 with an apparent pA2 value of 5.90 (Fig. 3; n = 6). As shown in Fig. 4, the effect of SG-86 was not modified by methylene blue (10- s tool/l), ethyl alcohol (0.4%) or glibenclamide (10 .5 mol/1). In the presence of these agents, the pD2 values for SG-86 were 1.90_+ 0.03, 1.93 _+ 0.03 and 1.85 _+ 0.02, respectively (n = 6 in each case).

Effects of SG-209 and nicorandil on cyclic GMP content Figure 5 depicts the changes in cyclic GMP content of isolated rabbit aorta produced by SG-209 (10-3mol/1) and nicorandil (3 x 10 .6 mol/1). The concentrations chosen were the ones that produced submaximal relaxation. The relaxation produced by 10 -3 tool/1 SG-209 was 78.7% _+ 3.4% (n = 6), while that by 3 x 10 -6 mol/1 nicorandil was 77.7% + 6.1% (n = 6) (Figs. 2 and 3). The changes in cyclic GMP content were followed for 10 min, as the phase of rapid relaxation by either agent occurred over this time interval. Over this period, the cyclic GMP content remained stable in the control group (no drug, n = 6 at each time point): cyclic GMP content before administration (0 min), after 1, 3 and 10 min of administration was 62.1 _ 7.1, 69.6 + 6.4, 61.2 + 9.5 and 56.8 _ 7.8 fmol/mg protein, respectively (n = 6 at each time point). SG-209 did not affect the cyclic GMP content at any of these time

238 200

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1 min

nml 3 rain

10 min

Fig. 5. Effect of nicorandil and SG-209 on the cyclic G M P content (cGMP) of the rabbit aorta. Concentrations that produce relaxations of around 80% of the maximum were chosen. Open column, control (no drug, n = 6 at each time point); cross-hatched column, SG-209-treated (10 - 3 mol/1, n = 6 at each time point); filled column, nicorandil-treated ( 3 x 1 0 - 6 m o l / 1 , n = 6 at each time point). *P < 0.05, **P < 0.01

points (n = 6 at each time point). On the other hand, nicorandil increased the cyclic G M P content markedly, effects which were significant after 3 and 10 min of the treatment (135.5 _ 11.6 and 143.7 +_ 16.2 fmol/mg protein, respectively; n = 6 in each case).

Discussion

SG-209 is a pyridine derivative in which the nitrate moiety of nicorandil (SG-75) has been replaced with an acetate residue. SG-209 dilates tracheal smooth muscle and increases tracheal arterial blood flow in anesthetized dogs with respective potencies of 1/20 and 1/10 compared with those of nicorandil (Maruyama et al. 1982). However, the mechanism of the smooth muscle relaxant effect of SG209 has not yet been elucidated. In the present study glibenclamide was shown to produce a parallel shift to the right of the relaxation concentration-response curve of SG-209, while methylene blue produced no such a shift. Furthermore, no increase in cyclic GMP content was observed on exposure of aortic segments to SG-209. These findings indicate that the opening of potassium channels was mainly responsible for the smooth muscle relaxant action of SG-209. In the present study methylene blue produced a shift to the right of the concentration-response curve for nicorandil. Furthermore, significant increases in tissue cyclic GMP content were observed. These findings are consistent with the idea that the in vitro smooth muscle relaxant activity of nicorandil is attributable to the presence of its nitrate moiety (Holzmann 1983; Kukovetz and Holzmann 1987; Eltze 1989) and is associated with an increased cyclic GMP formation, an action characteristic of smooth muscle relaxation by NO-forming vasodilators (Schultz et al. 1977; Kukovetz et al. 1979; Axelsson et a1.1981 ; Gruetter et al. 1981). However, membrane hyperpolarization by nicorandil was observed in early microelectrode studies conducted

with porcine and guinea-pig arteries (Furukawa et al. 1981). Subsequently, a decrease in membrane resistance and a cessation of on-going spontaneous electrical discharges were also reported (Furukawa et al. 1981; Itoh et al. 1981; Karashima et al. 1982; Inoue et al. 1983). These data, together with those provided by S6Rb efflux experiments in rat portal vein, guinea-pig trachea and taenia caeci (Allen et al. 1986; Weir and Weston 1986a, b) showed that nicorandil could also open membrane potassium channels. Furthermore, it was found that the hyperpolarizing effects of nicorandil in dog mesenteric artery and guinea pig trachea could be abolished by the potassium channel blocker, tetraethylammonium (TEA) (Inoue et al. 1983; Allen et al. 1986). Despite these observations, the contribution which potassium channel opening makes to the mechanical (relaxant) effects of nicorandil in smooth muscle is still unclear; for example, inhibition by potassium channel blockers has never been demonstrated as regards the relaxant effects of nicorandil (Allen et al. 1986; Eltze 1989). In the present study, a potent potassium channel blocker, glibenclamide (Cavero et al. 1989), failed to inhibit the relaxant effect of nicorandil in the absence of methylene blue. However, in the presence of methylene blue, an inhibitor of soluble guanylate cyclase, glibenclamide inhibited the relaxant effects of nicorandil (10 -5 mol/1 and above) (Fig. 2), suggesting that potassium channel activation contributes to the relaxant effects of high concentrations of nicorandil. This is an indirect but clear-cut demonstration that potassium channel opening activity is involved in smooth muscle relaxant activity of nicorandil. Glibenclamide failed to modify the concentration-response curve for nicorandil in the absence of methylene blue because the tissue was already maximally relaxed due to activation of guanylate cyclase by nicorandil ( < 10- 5mol/1). Only when guanylate cyclase activation by nicorandil was attenuated by methylene blue did activation of potassium channels become important and allow detection of the antagonistic effect of glibenclamide. The concentrations of nicorandil at which indications for potassium channel opening activity were observed in the present study corresponded to those shown to modulate membrane conductance or to increase S6Rb efflux (Furukawa et al. 1981; Itoh et al. 1981; Karashima et al. 1982; Inoue et al. 1983, 1984; Weir and Weston 1986a, b) and are in agreement with the conclusions of Allen et al. (1986) and Kreye et al. (1991) that relatively high concentrations of nicorandil are necessary to develop potassium channel opening activity. A recent in vivo study conducted in conscious SHR or in normotensive anesthetized rats (Richer et al. 1990) demonstrated that the decrease in blood pressure produced by nicorandil could be antagonized by glibenclamide. This discrepancy may be explained by a higher sensitivity of resistive vessels to potassium channel opening activity (Richer et al. 1990). Needless to say, the resistive vessels are more important for regulation of the blood pressure than the large conductive arteries. As the main metabolite (denitrated) of nicorandil, SG86, has no pharmacological activity (Sakai et al. 1980; Inoue et al. 1984), it is apparent that the presence of the

239 n i t r a t e g r o u p is essential for the actions of n i c o r a n dil. A l t h o u g h very high c o n c e n t r a t i o n s o f SG-86 ( > 1 0 - 3 mol/1) caused r e l a x a t i o n in the p r e s e n t study, this effect was n o t m o d i f i e d b y either m e t h y l e n e b l u e or glibenclamide, suggesting a non-specific a c t i o n at such high c o n c e n t r a t i o n s . I n view o f the f i n d i n g s o f the p r e s e n t study t h a t SG209, a c o m p o u n d w i t h o u t a n i t r a t e moiety, p r o d u c e d r e l a x a t i o n t h r o u g h p o t a s s i u m c h a n n e l activation, it m a y be c o n c l u d e d t h a t the b a s a l structure o f SG-86 plus a n a d d i t i o n a l g r o u p definitely o t h e r t h a n a h y d r o g e n alone m a y be the f u n d a m e n t a l structure for a c t i v a t i o n o f the p o t a s s i u m c h a n n e l in these p y r i d i n e derivatives.

Acknowledgement. The

authors wish to thank Chugai Pharmaceutical Co., Tokyo, Japan for generous supply of nicorandil, SG-209 and SG-86.

References

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