Potassium-channel activators act primarily to increase cellular potassium conductance by opening membrane potassium channels and consequently result in membrane hyperpolarization (1-3). It is hypothesized that hyperpolarization induced by the potassium-channel activators causes vasorelaxation by preventing the opening of voltage-activated calcium channels (4,5). Potassium-channel activators such as cromakalim, pinacidil, and nicorandil are known to reduce blood pressure in several experimental animals of hypertension because of a reduction in peripheral vascular resistance (6-9). Potassium-channel activators are also known as potent coronary vasodilators (10-12) and exert direct protective effects on ischemic myocardium in numerous models (13-16) independent of their peripheral or coronary vasodilator activities, thus offering potentially useful treatment for angina and myocardial ischemia (17,18).
SKP-450 (KR-30450), 2-(2″(1″,3″-dioxolan-2-yl) 2-methyl-4-(2′-oxopyrrolidin-1-yl) -6-nitro-2H-1-benzopyran (Fig. 1), is a benzopyran derivative closely related to lemakalim and was synthesized at the Korea Research Institute of Chemical Technology (KRICT, Taejon, Korea). It was known that SKP-450 antagonized the inhibitory effect of adenosine triphosphate (ATP) on the ATP-sensitive potassium-channel activity in single rat ventricular myocytes (19), probably by a mode of action that is distinct from those of lemakalim, pinacidil, and nicorandil (19,20). Preliminary results from studies with isolated tissue showed that the vasodilatory effect of SKP-450 was inhibited by glibenclamide, a selective blocker of ATP-sensitive potassium channels, suggesting that ATP-sensitive potassium channels were involved in the vasorelaxation of this compound. In our study, we examined the antihypertensive effects of SKP-450 in conscious spontaneously hypertensive rats (SHRs), renal hypertensive rats (RHRs), deoxycorticosterone acetate (DOCA)/salt hypertensive rats (DHRs), and normotensive rats (NRs) and characterized the hemodynamic profile of SKP-450 in anesthetized and conscious beagle dogs.
Antihypertensive effects in conscious rats
This study conformed with the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health. Male SH (12-14 weeks, Charles River, Yokohama, Japan), RH, DH, and normotensive Sprague-Dawley rats (300-350 g; KRICT) were used. RHRs and DHRs were prepared from Sprague-Dawley rats. For preparing RHRs, the left renal artery of rats was completely ligated under ketamine (125 mg/kg, i.p.) anesthesia (21,22). They were fed normal diet and water ad libitum for 1 week. For preparing DHRs, the animals were implanted with DOCA pellets (100 mg/kg) subcutaneously at the dorsal neck area under ketamine anesthesia. They were maintained on normal diet and 1% NaCl in tap water for 4 weeks. All rats were housed in plastic cages in rooms maintained on 12 h light-dark cycles.
The blood pressure was measured from the animals by a direct method as follows. The polyethylene catheter (PE-50) connected to PE-10 catheter filled with heparinized saline solution (20 IU/ml) was inserted into the femoral artery of animals under ketamine anesthesia, and the free end of catheter was passed through a subcutaneous tunnel to be exposed and fixed at the dorsal skin of the neck. The blood pressure was measured with pressure transducer (CDX-III; Modular Instruments, Malvern, PA, U.S.A.) coupled to a physiograph (Modular 8000 Signal processor), and HR was derived from the blood pressure pulse, both parameters being analyzed and stored by Biowindow program (Modular Instruments). The animals were allowed 1 day to recover and stabilize in individual cages. Mean arterial pressure (MAP) and HR were monitored for 24 h after oral administration of SKP-450 and lemakalim at single doses of 0.01, 0.03, 0.1, and 0.3 mg/kg. To assess the mechanism of reflex tachycardia produced by SKP-450, another series of experiments was conducted in conscious NRs pretreated with propranolol (20 mg/kg, i.v.) 15 min before SKP-450 (0.03 mg/kg, i.v.). The ability of glibenclamide to antagonize the hypotensive responses of cumulative SKP-450 (0.001-1 mg/kg, i.v.) also was evaluated in conscious NRs. Glibenclamide (20 mg/kg), at a dose comparable to that used by other investigators in rat (23), was intravenously injected 20 min before SKP-450 administration.
Hemodynamic profiles in anesthetized dogs
Male beagle dogs (Marshall Farms, NY, U.S.A.) weighing 8-12 kg were initially anesthetized with sodium pentobarbital (35 mg/kg) by intravenous injection into the left cephalic vein and maintained throughout the experiment under a continuous infusion of sodium pentobarbital (3-4 mg/kg/h) via a catheter fixed in the left cephalic vein. After tracheal intubation, artificial respiration was performed by a ventilator (dog ventilator 5025; Ugo Basile, Varese, Italy) with room air; the tidal volume and respiratory rate were adjusted to maintain pCO2 at 30-35 mm Hg. To measure the blood gas (blood-gas analyzing system 280; Ciba-Corning, Medfield, MA, U.S.A.), ∼0.5 ml of blood was withdrawn from the catheter inserted in brachial artery at regular intervals. Rectal temperature was monitored and maintained at 38 ± 1°C with a heating pad. Arterial pressure was measured with a Statham P23XL pressure transducer (Grass Instruments, Quincy, MA, U.S.A.) via a catheter filled with heparinized saline placed in the right femoral artery, and HR with a tachometer (Biotachometer; Gould Inc., Cleveland, OH, U.S.A.) triggered by the arterial pulse wave. A Millar Micro-Tip catheter (6F; Millar Instruments, Houston, TX, U.S.A.) was advanced into the left ventricle through the left carotid artery for the measurements of left ventricular systolic pressure (LVSP) and end-diastolic pressure (LVEDP; the LVBP at the end of the filling phase), LV dP/dtmax, and systolic time (ST; time that elapsed from LVEDP to LV end-systolic pressure). LV dP/dtmax was measured with a differentiator amplifier (Gould) triggered by LVP, and ST was read from the chart graph. To measure cardiac output (CO; L/min) with a thermodilution method and pulmonary arterial pressure, a thermistor-tipped catheter was introduced into the pulmonary artery through a jugular vein with the help of radiographs (EP1002; Ihwa X-ray Instruments, Seoul, Korea). Then the thermistor catheter was connected to a CO computer (Cardiomax II; Columbus Instruments, Columbus, OH, U.S.A.) to monitor CO, and the pressure catheter to a Statham P23XL pressure transducer to monitor pulmonary arterial pressure. All signals except CO were recorded continuously on a Gould 2000 chart recorder (Gould). The standard hemodynamic parameters derived in this study were tension-time index (TTI), calculated as HR × ST × LV end-systolic pressure/1,000; rate-pressure product (RPP; mm Hg × beats/min), computed as MAP × HR/1,000; and total peripheral resistance (TPR; mm Hg/L/min), calculated as MAP/CO (10). After all hemodynamic parameters had stabilized postoperatively for ≥40 min, SKP-450 was given intraduodenally at single doses of 0.003, 0.01, and 0.03 mg/kg (administration volume, 1 ml/kg) via a tube inserted and fixed into the caudal pylorus. Dogs were monitored for 4 h after drug administration.
Effects on arterial blood flow in anesthetized dogs
A separate group of anesthetized male beagle dogs were used to determine the effects of SKP-450 on arterial blood flow. The surgical procedures and the methods for measuring MAP and HR were the same as those already described. A left lateral thoracotomy was made at the fifth intercostal space, and the pericardium was opened and retracted. A Doppler-flow probe (Crystal Biotech, Northboro, MA, U.S.A.) was placed around the left circumflex coronary artery (LCX). Doppler-flow probes of suitable sizes also were placed around the left renal, superior mesenteric, and right femoral arteries, respectively. Coronary (CBF), renal (RBF), mesenteric (MBF) and femoral arterial blood flow (FBF) were measured with a Doppler flowmeter (CB1-8000; Crystal Biotech) connected to each flow probe and recorded on a chart recorder (MFE Instruments, Beverly, MA, U.S.A.), whereas other parameters were monitored by a Gould 2000 chart recorder (Gould). Vascular resistance (VR; mm Hg/ml/min) through individual vascular beds was calculated as MAP/BF. After dogs were allowed to recover stable cardiovascular performance, SKP-450 was given intraduodenally at single doses of 0.01 and 0.03 mg/kg via a tube inserted and fixed into the caudal pylorus in a volume of 1 ml/kg. Dogs were monitored for 4 h after drug administration.
Effects on blood pressure in conscious dogs
Male beagle dogs weighing 8-12 kg were anesthetized with sodium pentobarbital (30 mg/kg) by intravenous injection into the left cephalic vein. Under the aseptic conditions, the left femoral artery was cannulated with a special long-term catheter device filled with heparin (1,000 IU/ml). The catheter was exteriorized through a subcutaneous tunnel at the back of neck. After 2 days of recovery, a pocketed jacket was placed on the dogs (Daejong Co., Seoul, Korea), which were trained to stand. Arterial pressure was measured with a Grass P23XL pressure transducer (Grass) and continuously recorded on a Gould 2000 physiograph (Gould). HR was derived from the arterial pressure pulse by ECG/Biotacho amplifier module of the Gould 2000 physiograph. SKP-450 was orally given to each animal at a single dose of 0.03, 0.1, and 0.3 mg/kg in a volume of 1 ml/kg. Dogs were monitored for 4 h after drug administration.
SKP-450 and lemakalim were synthesized at Bio-Organic Science Division, KRICT. DOCA pellets containing deoxycorticosterone acetate, 100 mg, were purchased from Innovative Research of America (Sarasota, FL, U.S.A.). Sodium pentobarbital was purchased Hanlim Pharmaceutical Co. (Seoul, Korea) and ketamine hydrochloride from Yuhan Co. (Seoul, Korea). Propranolol hydrochloride and glibenclamide were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Propranolol was dissolved in 0.9% saline and glibenclamide in demethylformide (DMF) and diluted with saline to yield a given concentration (the final concentration of DMF, 10%). SKP-450 and lemakalim were suspended in 0.5% carboxymethylcellulose (CMC) for oral and intraduodenal administration and dissolved in 5% polyethyleneglycol 400 in saline for intravenous administration.
All values are expressed as mean ± SEM. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett's test for multiple comparisons (Sigma Stat; Jandel Co., San Rafael, CA, U.S.A.). In all the comparisons, the difference was considered to be statistically significant at p <0.05. The ED20 values (a dose that decreased MAP by 20%) were obtained from a linear regression of effects versus log dose.
Antihypertensive effects in conscious rats
The effects of the orally administered SKP-450 (0.01-0.3 mg/kg) on MAP and HR in various rat models of hypertension are shown in Fig. 2. The mean predose values of MAP and HR are shown in Table 1. In rats of all types studied, SKP-450 produced dose-dependent reductions in MAP with 2-5 min taken to exert its action and 20-30 min to reach the maximal effects. The antihypertensive effects of SKP-450 in SHRs lasted >6 h at all doses (p < 0.05), which was much longer than in RHRs (2-4 h), DHRs (2-4 h), and NRs (4-6 h). The calculated ED20 values of SKP-450 in SHRs, RHRs, and DHRs were smaller than those in NRs (0.021 ± 0.003 mg/kg, 0.013 ± 0.003 mg/kg, 0.024 ± 0.009 mg/kg, and 0.034 ± 0.003 mg/kg, respectively; p < 0.05 for SHRs and RHRs vs. NRs), indicating that the antihypertensive effects of SKP-450 were more potent in hypertensive rats than in NRs.
At a low dose of 0.03 mg/kg, SKP-450-induced maximal decreases from baseline MAP values in SHRs, RHRs, and DHRs (20.6 ± 2.6%, 32.2 ± 7.3%, and 26.0 ± 8.0%, respectively,) were larger than those in NRs (15.6 ± 1.1%), although statistical significance was not noted, whereas at a higher dose of 0.1 mg/kg, SKP-450-induced maximal decrease in DHRs (55.8 ± 1.6%; p < 0.05) were significantly larger than those in SHRs, RHRs, and NRs (30.8 ± 1.9%, 46.4 ± 4.2%, and 41.9 ± 2.1%, respectively). The decrease in MAP was accompanied by a dose-dependent increase in HR. In SHRs, tachycardiac effects reached the maximum between 10 and 20 min after SKP-450 administration (Emax at 0.1 and 0.3 mg/kg: 39.9 ± 7.3% and 41.3 ± 3.0%, respectively; p < 0.05 vs. control) and lasted for 2-4 h at significantly increased level.
However, at lower doses of SKP-450 (0.01 and 0.03 mg/kg), the tachycardiac effect in SHRs was not significant despite the significant decrease in MAP. In RHRs, the significant tachycardiac effect was induced at the highest dose only, with a relatively short duration of 1.5 h (Emax at 0.3 mg/kg, 22.4 ± 4.8%; p < 0.05 vs. control), but it was negligible at lower doses of SKP-450 (0.01, 0.03, and 0.1 mg/kg) despite the significant decrease in MAP, indicating the weakest tachycardiac effect in RHRs among all kinds of rats. In DHRs, tachycardiac effects reached the maximum between 10 and 20 min after SKP-450 administration (Emax at 0.1 mg/kg, 44.2 ± 8.2%; p < 0.05 vs. control) and persisted for 2 h at a significant level. However, at a lower dose of SKP-450 (0.03 mg/kg), the significant tachycardiac effect was not noted in DHRs despite the significant decrease in MAP. In NRs, SKP-450 produced a dose-dependent increase in HR with a maximal increase occurring between 10 and 20 min after the dose (Emax at 0.03, 0.1, and 0.3 mg/kg: 20.9 ± 2.0%, 52.6 ± 3.8%, and 57.8 3.4%, respectively; p < 0.05 vs. control), and the tachycardiac effect persisted for the duration of hypotension (1, 2-4, and >6 h at three doses, respectively). The tachycardiac effects of SKP-450 were more potent in NRs than in other types of hypertensive rats studied, as evidenced by induction of significant tachycardia at all doses in NRs and induction of insignificant tachycardia at lower doses in hypertensive rats with longer duration of tachycardia in NRs. These results suggest the differential effects of SKP-450 on HR in hypertensive and normotensive rats.
The changes in MAP and HR in various rat models of hypertension after oral administration of lemakalim are shown in Fig. 3. The mean predose values of MAP and HR are shown in Table 1. In rats of all types studied, lemakalim produced significant dose-dependent reductions in MAP with a maximal decrease occurring between 10 and 20 min after the dose, these antihypertensive effects lasting ∼2 h in all types of rats except the highest dose in SHRs (>6 h). Lemakalim was significantly more potent in RHRs and DHRs than in NRs (ED20 values, 0.018 ± 0.002 mg/kg, 0.016 ± 0.006 mg/kg, and 0.063 ± 0.009 mg/kg, respectively; p < 0.05 vs. NRs), but it was significantly less potent in SHRs (ED20 values, 0.107 ± 0.009 mg/kg; p < 0.05) compared with other types of rats studied. On the other hand, in SHRs and NRs, SKP-450 was about fivefold and twofold more potent than lemakalim, respectively, in decreasing MAP; in RHRs and DHRs, however, it was similar to lemakalim. At 0.03 mg/kg of lemakalim, the maximal decrease of baseline MAP was significantly greater in RHRs and DHRs (30.4 ± 8.5% and 29.9 ± 3.2%, respectively; p < 0.05) than in SHRs and NRs (7.5 ± 2.4% and 13.5 ± 1.6%, respectively), and at 0.1 mg/kg of SKP-450, the maximal decrease was also significantly greater in RHRs and DHRs (48.7 ± 2.1% and 48.9 ± 2.4%, respectively; p < 0.05) than in SHRs and NRs (17.7 ± 3.0% and 30.2 ± 2.4%, respectively). In SHRs, the decrease in MAP was accompanied by dose-dependent increase in HR, which reached the maximal effect within 10 min after administration of each dose of lemakalim (Emax at 0.1 and 0.3 mg/kg, 30.3 ± 3.7% and 32.0 ± 7.1%, respectively; p < 0.05 vs. control) and lasted for 1 h at significantly increased level. The tachycardiac effect in RHRs reached the maximum within 10 min after administration of lemakalim (Emax at 0.03, 0.1, and 0.3 mg/kg: 27.2 ± 2.4%, 43.0 ± 8.1%, and 33.0 ± 11.5%, respectively; p < 0.05 vs. control), and persisted for 1 h at significantly increased level. In DHRs, tachycardiac effects reached the maximum within 10 min after lemakalim administration (Emax at 0.1 mg/kg: 46.6 ± 7.2%; p < 0.05 vs. control) and persisted for 1 h. However, at a lower dose of lemakalim (0.03 mg/kg), the significant tachycardiac effect was not noted in DHRs despite the significant decrease in MAP. The tachycardiac effects of lemakalim were more potent in NRs than in other types of hypertensive rats studied: the maximal effects at 0.1 and 0.3 mg/kg were 47.6 ± 4.4% and 54.3 ± 5.8% (p < 0.05 vs. control), respectively, and the tachycardiac effects persisted for the duration of hypotension (2 h).
Propranolol (2 mg/kg, i.v.), a β-adrenoceptor blocker, caused a slight reduction in HR in NRs (from 317 ± 12 beats/min to 280 ± 7 beats/min; n = 5; p < 0.05) with minimal effects on MAP (from 101 ± 6 mm Hg to 106 ± 7 mm Hg). Effective β-blockade by propranolol at the dose used was evidenced by the failure of isoprenaline (1 μg/kg, i.v.) to increase HR (38.0 ± 3.9% compared with 0.7. ± 2.6%; n = 5; p < 0.05) in atropine-treated (2 mg/kg) rats. The pretreatment with propranolol prevented the tachycardiac response to SKP-450 (0.03 mg/kg, i.v.; 40.9 ± 3.9% compared with 10.4 ± 2.7%; n = 5; p < 0.05) without affecting the antihypertensive response (33.4 ± 0.8% compared with 38.3 ± 2.7%).
In the experiments with glibenclamide, it was shown that pretreatment with glibenclamide (20 mg/kg, i.v.), a selective ATP-sensitive potassium-channel blocker, did not significantly change baseline MAP and HR in NRs (MAP, from 97 ± 4 to 107 ± 2 mm Hg; HR, from 312 ± 2 to 300 ± 12 beats/min) but significantly antagonized the antihypertensive effects of intravenously administered SKP-450 (ED20 values, from 0.008 ± 0.001 to 0.039 ± 0.006 mg/kg; p < 0.05, at doses of 0.003, 0.01, and 0.03 mg/kg (Fig. 4). The tachycardiac response to SKP-450 was also significantly antagonized by glibenclamide (from 24.2 ± 4.6% and 42.4 ± 4.8% to 2.1 ± 3.3% and 16.5 ± 7.9% at 0.01 and 0.03 mg/kg, respectively; p < 0.05).
Hemodynamic effects in anesthetized dogs
The effects of intraduodenally administered SKP-450 on hemodynamic parameters, including those for LV function, in anesthetized beagle dogs are shown in Fig. 5. The mean predose values of hemodynamic parameters are shown in Table 2. SKP-450 (0.003, 0.01, and 0.03 mg/kg, i.d.) decreased MAP and TPR in a dose-related manner with a rapid onset of action (2-5 min), the maximal effects being reached within 15 min. The ED20 value for SKP-450 in decreasing MAP was 0.007 ± 0.002 mg/kg. HR was not changed by SKP-450; at all doses tested, it was dose-dependently increased, and positive dP/dtmax was slightly increased by SKP-450 at all doses tested except a transient decrease at the highest dose. SKP-450 dose-relatedly decreased three indirect indexes of myocardial oxygen demand: RPP, TTI, and ST. In these experiments, the reduction of RPP was exclusively caused by a decrease of LVP because HR was not affected by SKP-450. However, SKP-450 also significantly shortened ST. Thus SKP-450-induced reduction in ST, together with the decrease in LVP, contributed to a reduction in TTI.
Effects on arterial blood flow in anesthetized dogs
The effects of intraduodenal administration of SKP-450 on blood flow and vascular resistance through coronary, renal, mesenteric, and femoral arteries in anesthetized beagle dogs are shown in Fig. 6. The mean predose values of blood flow and vascular resistance from each artery are shown in Table 2. SKP-450 increased CBF by 17.6 ± 6.0% and 276.5 ± 47.2%, while decreasing CVR by 29.6 ± 6.9% and 83.7 ± 3.2% at 0.01 and 0.03 mg/kg, respectively. It took ∼2-5 min for SKP-450 to exert its action, and the effects of SKP-450 on CBF and CVR lasted >4 h at the higher dose. RBF was gradually increased with time by SKP-450 at a dose of 0.01 mg/kg (maximum, 21.5 ± 9.4%); at a dose of 0.03 mg/kg, however, RBF was decreased (maximum, 42.0 ± 10.0; p < 0.05) and then gradually increased over baseline (maximum, 27.3 ± 4.3%). RVR was not significantly decreased by SKP-450. MBF was increased by 20.9 ± 22.5% and 42.2 ± 22.4% at 0.01 and 0.03 mg/kg (p < 0.05), respectively, and MVR was decreased dose-relatedly (maximum, 21.7 ± 4.0% and 56.8 ± 10.9%; p < 0.05, respectively). SKP-450 increased FBF at a dose of 0.03 mg/kg without statistical significance, and FVR was significantly reduced by SKP-450 at a dose of 0.03 mg/kg (maximum, 38.2 ± 11.2%; p < 0.05).
Effects on blood pressure in conscious dogs
The effects of orally administered SKP-450 on MAP and HR in conscious beagle dogs are shown in Fig. 7. The mean predose values of MAP and HR were 113 ± 4 mm Hg and 111 ± 4 beats/min, respectively. As in anesthetized dogs, SKP-450 dose-dependently decreased the MAP, with a rapid onset of action (3-10 min), and reached its maximal effects within 15 min. The ED20 values for SKP-450 were 0.029 ± 0.003 mg/kg, and its maximal hypotensive effects at doses of 0.03, 0.1, and 0.3 mg/kg were 19.6 ± 2.3%, 43.6 ± 5.9%, and 54.3 ± 4.6%, respectively. The duration of the antihypertensive effect of SKP-450 lasted 1 h at a dose of 0.03 mg/kg and 2-2.5 h at doses of 0.1 and 0.3 mg/kg. In contrast to its effects in anesthetized dogs where no effects on HR were noted, however, SKP-450 increased HR in a dose-related manner in conscious dogs, with the maximal tachycardia occurring between 10 and 30 min at doses of 0.003 and 0.01 mg/kg, and 1.5 h at a dose of 0.3 mg/kg (Emax, 34.2 ± 13.4%, 79.1 ± 18.4%, and 94.4 ± 12.4%, respectively; p < 0.05 vs. control) and significantly lasted for the duration of hypotension. The increase in HR was presumably related to reflex sympathetic activation or vagal withdrawal or both.
Our results indicate that SKP-450, a structurally novel potassium-channel activator, is an orally active and potent coronary and peripheral vasodilator in vivo. We used three rat models of hypertension: the Okamoto strain of SHRs, a widely recognized model of essential hypertension; two-kidney, one-ligated RHRs, a high-renin model; and DHRs, a low-renin model of volume-dependent hypertension. SKP-450 produced a dose-dependent decrease in MAP in all kinds of hypertensive rats studied. On the basis of the ED20 values, the potency order for the hypotensive activity of SKP-450 was RHRs > SHRs = DHRs > NRs, which indicates that hypertensive rats are more sensitive to SKP-450 than are normotensive rats. The hypotensive effects of SKP-450 lasted for ∼4 h in RHRs, DHRs, and NRs, and >6 h, particularly in SHRs. The longer duration of oral SKP-450 at all doses used in SHRs than in other rat models of hypertension may be important feature in developing this compound as a new antihypertensive agent. The tachycardiac effects of SKP-450 were more potent in NRs than in various rat models of hypertension. Furthermore, in various rat models of hypertension, SKP-450, especially at lower doses, did not induce tachycardia despite the significant decrease in MAP. These findings suggest differential effects of SKP-450 on HR in hypertensive and normotensive rats.
The potency order of hypotensive effects of lemakalim was RHRs = DHRs > NRs > SHRs, which indicated that lemakalim was more potent in RHRs and DHRs than in SHRs and NRs. In SHRs and NRs, SKP-450 was fivefold and twofold more potent than lemakalim in decreasing MAP, respectively; in RHRs and DHRs, however, it was similar to lemakalim. Intravenous lemakalim was reported to display a greater hypotensive action in SHRs than in NRs (24). However, in our experiments, the orally administered lemakalim was equipotent between SHRs and NRs or less potent in SHRs than NRs, confirming recently published results obtained under similar experimental conditions (23,25).
SKP-450, repeatedly given to SHRs over 21 days (0.01 and 0.03 mg/kg, p.o., once daily), did not alter the degree and pattern of its antihypertensive effects and the reactivity of isolated aorta to various vasoconstrictors and vasodilators (unpublished data), suggesting that long-term treatment with SKP-450 does not develop the tolerance to antihypertensive activity and change in vascular reactivity.
Moreover, glibenclamide, a specific blocker of ATP-sensitive potassium channels, significantly antagonized the antihypertensive effects of SKP-450 in conscious NRs, indicating the mediation of the effects of SKP-450 via this channel. In all kinds of hypertensive rats tested, SKP-450 and lemakalim produced an increase in HR of a similar magnitude. As previously reported for other potassium-channel activators (23,26), a β-adrenoceptor blocker, propranolol, prevented this effect of SKP-450 without affecting the hypotensive response, suggesting that it was probably of reflex origin and represents a hemodynamic counterregulation of the decrease in MAP rather than a direct action of SKP-450 on the heart.
In anesthetized dogs, the most important hemodynamic effects of intraduodenally administered SKP-450 were dose-dependent decreases in MAP. This effect is mainly on its arterial vasodilator action, leading to a decrease in TPR. SKP-450 produced a strong, significant increase in CBF at a dose of 0.03 mg/kg, although this was negligible at a dose of 0.01 mg/kg; SKP-450 exerted much more potent and longer-lasting effects on CBF than on any other parameters including MAP. SKP-450 significantly decreased CVR, even at a lower dose at which no change in CBF was noted. Moreover, intravenously administered SKP-450 (0.001-0.01 mg/kg) in anesthetized dogs increased CBF markedly and dose-dependently, even at a low dose that did not exhibit any hypotensive effect (unpublished data). A decrease in myocardial oxygen demand as well as a decrease in CVR would be also helpful for the relief from angina, particularly angina involving vasospasm. The myocardium oxygen demand could be reduced by potassium-channel activators, which produce a decrease in afterload together with some negative inotropic effect. Indirect measures of myocardial oxygen demand such as RPP, TTI, and ST were dose-dependently decreased by SKP-450, suggesting that myocardial oxygen demand was reduced. However, these indirect data could be misleading because myocardial oxygen demand is a complex function of afterload, preload, HR, contractile state, and wall tension (27). Potassium-channel activators also affect myocardial tissue, as reflected in the negative inotropic effects and the shortening of the cardiac action potential in isolated tissues (28,29) occurring at doses higher than those that have significant vascular actions. In our study, SKP-450 slightly increased positive dP/dtmax at low doses (0.003 and 0.01 mg/kg) but transiently decreased that at a higher dose (0.03 mg/kg) in anesthetized dogs. This transient decrease in positive dP/dtmax at a higher dose may result from the markedly decreased afterload, because positive dP/dtmax is known to be highly sensitive to changes in afterload (10). However, we could not still rule out the possibility that the decrease in positive dP/dtmax is caused not only by a decrease in afterload but also by a direct action on cardiomyocytes. The latter possibility is supported by the findings that some potassium-channel activators significantly reduce right ventricular contractile force in dogs, assessed in vivo with a strain-gauge transducer (10) under the condition of direct measurement of myocardial contractile force independent of afterload. Experimental evidence shows that potassium-channel activators can preserve the ischemic myocardium, improve the function of the stunned myocardium, and reduce mortality after myocardial infarction (30,31). Thus SKP-450 and other potassium-channel activators can potentially be useful in the treatment of coronary artery disease. SKP-450 produced a marked reduction in RBF in anesthetized dogs without significant changes in RVR. The reduction in RBF induced by SKP-450 could be explained in part by direct activation of the renin-angiotensin system, as suggested for potassium-channel activators (31,32). The effects of SKP-450 on RVR were similar to those of some other potassium-channel activators including Ro 31-6930 (26), nicorandil, and pinacidil (33), that were not reported to reduce RVR, although cromakalim significantly decreased RVR in the anesthetized dogs (33). Thus our findings and those of others may suggest that the effects on RVR are not generalized for all the potassium-channel activators. On the other hand, the effects of SKP-450 on mesenteric and femoral artery were similar to those of cromakalim, which induced no change in MBF and FBF and a large decrease in MVR and FVR (33).
The orally administered SKP-450 in conscious beagle dogs produced a dose-dependent decrease in MAP, and its ED20 values for decrease in MAP were similar with those for conscious NRs (ED20 values, 0.030 ± 0.009 vs. 0.034 ± 0.003 mg/kg, respectively). SKP-450 in dogs under anesthesia was about threefold more potent than in conscious dogs, and this might be ascribed to differences in administration route (oral vs. intraduodenal administration) or changes in nervous control of cardiovascular function under anesthesia or both. Unlike its effects in anesthetized dogs, SKP-450 concurrently increased HR in a dose-related manner in conscious dogs, which might be the result of reflex autonomic changes resulting from peripheral vasodilation. It was also often shown that potassium-channel activators decrease peripheral resistance and produce reflex increases in HR in conscious animals (23,26). Thus a significant increase in HR occurring during long-term use of potassium-channel activators in humans would limit the other beneficial effects these compound may have, even when it is possible to attenuate such effects by developing controlled-release formulations. Accordingly, it is unlikely that potassium-channel activators could be used as monotherapy for patients with hypertension, and if so, their antihypertensive efficacy could be optimized when given in combination with other types of antihypertensive agents such as diuretics, angiotensin-converting enzyme inhibitors, angiotensin II-receptor antagonists, and β-adrenoceptor-blocking agents.
Our results demonstrated that SKP-450 is an orally active, potent potassium-channel activator that reduces arterial blood pressure in anesthetized and conscious beagle dogs and in various models of conscious hypertensive rats. In addition, it appears that SKP-450 significantly increases CBF without having direct negative inotropic actions at general antihypertensive doses, and thus SKP-450 could be potentially useful for the treatment of hypertension and certain forms of angina.
Acknowledgment: We thank Mr. Ho Won Seo for his excellent technical assistance. This work was supported in part by a grant from The Ministry of Science and Technology, Korea.
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