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ANTI-ULCER ACTIVITY OF SKP-450, A NOVEL POTASSIUM CHANNEL ACTIVATOR, IN RATS
Pharmacological Research, Vol. 40, No. 3, 1999 Article No. phrs.1999.0512, available online at http:rrwww.idealibrary.com on
ANTI-ULCER ACTIVITY OF SKP-450, A NOVEL POTASSIUM CHANNEL ACTIVATOR, IN RATS HYAE GYEONG CHEON a , HYUN JUNG KIM a , HYE KYOUNG MO a , SUNG EUN YOO b and BYUNG HO LEE a,U a
Screening and Toxicology Research Center, and bBio-Organic Science Di¨ ision, Korea Research Institute of Chemical Technology, a100, Jang-dong, Yusong, Taejon 305-343, Korea Accepted 9 March 1999
The anti-ulcer effects of SKP-450, a new potassium channel activator, were evaluated on basal and histamine-induced gastric acid secretion, and against experimentally-induced ulcers such as ethanol-induced and NaOH-induced gastric ulcers. In the pylorus-ligated rat, SKP-450 Ž0.1]0.5 mg kgy1 . significantly decreased volume and concentration of gastric juice, and total acid output ŽED50 : 0.12 mg kgy1 .. SKP-450 Ž0.3]3.0 mg kgy1 . also inhibited histamine-induced gastric acid secretion, maximal effects being achieved at 1.0 mg kgy1 Ž37.9% inhibition.. In the 95% ethanol-treated rats, SKP-450 significantly reduced the mucosal lesions Ž46.9 and 31.4% inhibition at 0.1 and 0.2 mg kgy1 , respectively.. A significant reduction in the ulcer index by SKP-450 was also observed in 0.3 N NaOH-treated rats Ž31.5 and 64.3% inhibition at 0.5 and 1.0 mg kgy1 , respectively.. The effects of SKP-450 on histamine-induced acid secretion and on NaOH-induced ulcers were inhibited by glibenclamide Ž20 mg kgy1 , i.v.., a selective blocker of ATP-sensitive potassium channel. These results indicate that SKP-450 possesses anti-ulcer effects and its effects may be mediated by activation of ATP-sensitive potassium channels. Q 1999 Academic Press KEY
WORDS:
SKP-450, potassium channel activator, anti-ulcer.
INTRODUCTION SKP-450 ŽKR-30450, Fig. 1. is a potent, orally active coronary and peripheral vasodilator in rats and dogs w1]3x, and is under clinical trials. It was reported that vasorelaxant effects of SKP-450 on rat aorta and its antihypertensive effects in rats and dogs were inhibited by glibenclamide, a selective blocker of ATP-sensitive potassium channel, suggesting the involvement of ATP-sensitive potassium channel in the effects of this compound w1, 2x. In addition, SKP-450 antagonized the inhibitory effect of ATP on the ATP-sensitive potassium channel activity in single rat ventricular myocytes w4x, probably via a mode of action that was distinct from those of ATP-sensitive potassium channel activators } lemakalim, pinacidil and nicorandil w4, 5x. Potassium channel activators including SKP-450 act primarily to increase cellular potassium conductance by opening membrane potassium channels, and consequently result in membrane hyperpolarizaU
Corresponding author.
1043]6618r99r090243]06r$30.00r0
tion w6, 7x. The hyperpolarization induced by potassium channel activators is believed to result in an inhibition of Ca channels w6x, a reduction of Ca2q release from intracellular Ca2q stores through the inhibition of inositol-1,4,5-triphosphate production w8x, and a reduction in sensitivity of contractile proteins to Ca2q w9x. This change of calcium flux due to activating potassium channels might influence the gastric secretion. Recent studies also revealed that the activation of ATP-sensitive potassium channels increases gastric mucosal blood flow w10x, and ATPsensitive potassium channels play an important role in protecting the gastric mucosa w11]13x. Peptic ulcers appear to result from overproduction of gastric acid andror decrease in gastric mucosal protective mechanisms. Consequently, reduction of gastric acid production as well as reinforcement of gastric mucosal protection have been main approaches for peptic ulcer therapy. Based on the previous reports that SKP-450 may induce potassium channel activation w1, 2, 4x, SKP-450 was postulated to exert an anti-ulcer activity by protection of gastric mucosa andror by inhibition of gastric secretion. Accordingly, in the present study, the effects of Q 1999 Academic Press
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Fig. 1. Chemical structure of SKP-450 ŽKR-30450..
SKP-450 have been evaluated on the experimentally-induced ulcers elicited by ethanol or NaOH. In addition, the effect of SKP-450 on the gastric acid secretion was examined.
MATERIALS AND METHODS
Inhibition of basal acid secretion in the pylorus-ligated rat This study conformed with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health. Gastric anti-secretory activity was determined by Shay’s method with some modification w14x. Male Sprague]Dawley ŽS.D.. rats Ž6 weeks, 180]250 g, Korea Research Institute of Chemical Technology, KRICT, Taejon, Korea. were fasted for 24 h before experiments. The pylorus of animals was ligated under diethylether anaesthesia, and then SKP-450 Ž0.1]0.5 mg kgy1 . was intraduodenally administered. The animals were allowed to recover and stabilize in individual cages. At 5 h after surgery, the rats were killed by cervical dislocation. Following isolation of the stomach, gastric juice was collected, centrifuged at 5000 rpm for 10 min, and then measured for volume. An aliquot was used for determination of acid concentration by titration with 0.1 N NaOH to an endpoint of pH 7.0 using the Orion 960 autochemistry analyser ŽBoston, MA, USA.. Acid output was calculated by multiplying the volume of gastric juice with the acid concentration.
Inhibition of histamine-stimulated gastric acid secretion For the lumen-perfused rat studies, the method of Ghosh and Schild w15x was used. Male S.D. rats Ž10 weeks, 300]350 g. were fasted for 24 h and anaesthetized with urethane Ž1.2 g kgy1 . by an intraperitoneal injection. After tracheal intubation, a polyethylene tube ŽPE 50. was inserted and fixed in the forestomach through the oesophagus, and another cannula Ži.d. 2]3 mm. was inserted into the pyloric region of the stomach through duodenum. Animals were perfused with 0.9% wrv NaCl solution Ž378C.
by using an infusion pump at a flow rate of 1.5 ml miny1 . The perfusate from the duodenum tube was collected with fraction collector Žmodel 2110, BioRad, Richmond, CA, USA. at 15-min intervals, and its volume and acid concentration were determined by using an Orion 960 autochemistry analyser. Rectal temperature was monitored and maintained at 37 " 18C with heating pad and lamp. After baseline had been stabilized postoperatively for at least 20 min, SKP-450 Ž0.3]3.0 mg kgy1 . was given intraduodenally. Histamine Ž2 mg kgy1 . in 0.9% wrv NaCl was intramuscularly injected as a secretagogue 30 min after the administration of SKP-450. The perfusate was collected for 2.5 h after the administration of the compound. Percent inhibition was calculated from the comparisons of area under the curve of acid output in the presence or absence of SKP-450. In a separate experiment, the ability of glibenclamide, a blocker of ATP-sensitive potassium channels, to antagonize the effects of SKP-450 on histamine-stimulated acid secretion was evaluated in rats. Glibenclamide Ž20 mg kgy1 ., at a dose comparable to that used by other investigators in rats w16x, was intravenously injected 20 min prior to SKP-450 administration.
Effects of SKP-450 on experimental ulcer models Gastric mucosal lesions elicited by ethanol or NaOH were produced according to the method of Robert et al. w17x. Male S.D. rats Ž6 weeks, 180]250 g. were fasted for 24 h prior to the experiments. SKP-450 Ž0.1]3.0 mg kgy1 . was orally given 1 h before oral administration of either 95% ethanol Ž1 mlrrat. or 0.3 N NaOH Ž1 mlrrat.. One hour later, the animals were killed by diethylether anaesthesia, and the stomachs were isolated. After fixing the stomach in 13 ml of 1% formalin for 1 h, the greater curvature of the stomach was opened. The lengths of macroscopical lesion were measured, summed up and compared with the group treated with either 95% ethanol alone or 0.3 N NaOH alone. All the group allocations of the experimental animals were done in a randomized order and under blinded conditions. In a separate experiment, the ability of glibenclamide to antagonize the effects of SKP-450 on NaOH-induced ulcer model was evaluated in rats. Glibenclamide Ž20 mg kgy1 ., was intravenously injected 20 min prior to SKP-450 administration.
Drugs SKP-450 and omeprazole were synthesized at Bio-Organic Science Division, KRICT. Glibenclamide was purchased from Sigma Chemical Co. ŽSt. Louis, MO, USA.. Carboxy methyl cellulose ŽCMC. was obtained from Showa Chemical Co. ŽTokyo, Japan.. Absolute ethanol was obtained from Hayman Ltd. Co. ŽEssex, UK.. Formalin Ž17%. was
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anti-secretory effect in ¨ i¨ o, SKP-450 was given intraduodenally to pylorus-ligated rats, and its effect on gastric secretion was examined. As shown in Table I, pylorus ligation for 5 h caused the accumulation of volume of gastric juice Žcontrol rats: 5.9" 0.5 ml 200 gy1 ., the increases in the acidity of the gastric juice and the total acid output Žcontrol rats: 110 " 2.8 m Eq mly1 and 661 " 64.4 m Eq 5 hy1 , respectively.. SKP-450 Ž0.1]0.5 mg kgy1 . significantly decreased the volume and the acidity of gastric juice in a dose-dependent manner Ž1.2" 0.3 ml 200 gy1 and 64.8" 8.9 m Eq mly1 at 0.5 mg kgy1 , respectively.. These reductions in both the volume and the acidity of gastric juice resulted in the decrease of total acid output. The total acid output was dose-dependently inhibited by SKP-450 at all doses tested in this experiment Ž85.9" 25.6 m Eq 5 h at 0.5 mg kgy1 , 87.0% inhibition ¨ s control.. The calculated ED50 value for SKP-450 was 0.12 mg kgy1 . Omeprazole, a known anti-secretory compound, caused a significant reduction in acid output at 3.0 mg kgy1 Ž323 " 42.9 m Eq 5 hy1 , 51% inhibition ¨ s control..
obtained from Merck Co. ŽDarmstadt, Germany.. Polyethylene glycol ŽPEG. 400 and urethane were obtained from Junsei Chemical Co. ŽTokyo, Japan.. Diethylether was obtained from Oriental Chemical Industry ŽSeoul, Korea.. SKP-450 was suspended either in 20% PEG 400 Žfor intraduodenal administration. or in 0.5% CMC Žfor oral administration.. Glibenclamide was dissolved in dimethylforamide ŽDMF. and diluted with 0.9% wrv NaCl to yield a given concentration Žthe final concentration of DMF, 20%..
Statistical analysis
All values are expressed as mean " SEM. Data were analysed by unpaired Student’s t-test and oneway analysis of variance followed by Dunnett’s test for multiple comparisons ŽSigma Stat, Jandel Co., San Rafael, CA, USA.. In all comparisons, the difference was considered to be statistically significant at P- 0.05. The ED50 values Ža dose that inhibited basal acid secretion by 50%. were obtained from a linear regression of effects ¨ s log dose.
RESULTS
Inhibition of histamine-stimulated gastric acid secretion
Inhibition of basal acid secretion in the pylorus-ligated rat
The volume of gastric content from duodenum tube was always regular and time-dependent in the anaesthetized stomach lumen perfused rat models,
In order to determine whether SKP-450 has an
Table I Effect of SKP-450 on gastric secretion in pylorus-ligated rats Treatment
Control SKP-450 SKP-450 SKP-450 Omeprazole
Dose (mg kgy 1)
Volume of gastric juice (ml 200 gy 1)
Acidity of gastric juice (m Eq mly 1)
Total acid output (m Eq 5 hy 1)
] 0.1 0.2 0.5 3.0
5.9" 0.5 2.8" 0.5U 2.1" 0.4U 1.2" 0.3U 3.6" 0.5
110 " 2.8 157 " 5.8 78.5" 18.8 64.8" 8.9U 90.8" 8.1
661 " 64.4 446 " 47.9 Ž32.5.U 165 " 42.5 Ž75.0.U 85.9" 25.6 Ž87.0.U 323 " 42.9 Ž51.1.U
Each values represent the mean " SEM Ž n s 5.; values in parenthesis mean percent inhibition; U P- 0.05 compared with control group.
Table II Effects of SKP-450 on acid output in the lumen-perfused rat stomach under histamine stimulation Treatment Control Žhistamine acid phosphate, i.m.. SKP-450 SKP-450 SKP-450 SKP-450 SKP-450 Ž1.0 mg kgy1 . qGlibenclamide Ž20 mg kgy1 . Omeprazole
Dose (mg kgy 1)
Total acid output (m Eq 150 miny 1)
] 0.3 0.6 1.0 3.0
562 " 16.3 421 " 10.2 Ž25.1.U 357 " 32.7 Ž36.5.U 349 " 6.1 Ž37.9.U 478 " 33.5 Ž14.9.U
] 1.0
468 " 26.5† 232 " 181 Ž58.7.
Data represent the mean " SEM Ž n s 6.; values in parenthesis mean percent inhibition; U P- 0.05 compared with control group; †P- 0.01 compared with 1 mg kgy1 of SKP-450.
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Table III Effects of SKP-450 on the ethanol-induced gastric lesions Treatment Control SKP-450 SKP-450 SKP-450 SKP-450 Omeprazole Omeprazole
Dose (mg kg y 1)
Ulcer index (mm)
] 0.1 0.2 0.3 1.0 30 100
48.8" 4.3 25.9" 3.2 Ž46.9.U 33.5" 1.0 Ž31.4.U 57.1" 2.1 Žy17.0. 57.9" 6.2 Žy18.6. 44.2" 12.3 Ž0.9. 12.6" 10.4 Ž74.1.U
Data represent the mean " SEM Ž n s 5.. Values in U parenthesis mean % inhibition. P - 0.05 compared with control group.
Fig. 2. Effects of SKP-450 on acid output in the lumenperfused rat stomach under histamine stimulation. Vehicle Ž`., 0.3 Žv., 0.6 Ž^., 1.0 Ž'. and 3.0 mg kgy1 ŽI. of SKP-450. Data represent the mean " SEM Ž n s 6.. U P 0.05 compared with control group.
and the administration of histamine Ž2 mg kgy1 , i.m.. in the rats had shown a reproducible acid-output profile to time ŽFig. 2.. The total acid output in control rats was 562 " 16.3 m Eq over a 150-min time period. SKP-450 Ž0.3]3.0 mg kgy1 . inhibited the total acid output in a biphasic manner ŽTable II.. SKP-450 reached its maximal effect at 1.0 mg kgy1 Ž349 " 6.1 m Eq, 37.9% inhibition ¨ s control. and its effect was reduced at the higher dose used Ž478 " 33.5 m Eq at 3.0 mg kgy1 , 14.9% inhibition ¨ s control.. Glibenclamide Ž20 mg kgy1 , i.v.. significantly blocked the inhibitory effect of SKP-450 on histamine-stimulated acid secretion Ž468 " 26.5 m Eq, P- 0.01 ¨ s 1.0 mg kgy1 of SKP-450.. For the reference, omeprazole administration Ž1.0 mg kgy1 . resulted in the considerable inhibition of total acid output Ž232 " 181 m Eq, 58.7% inhibition ¨ s control..
Effects of SKP-450 on experimental ulcer models Oral administration of 95% ethanol or 0.3 N NaOH produced severe band-like mucosal haemorrhage in the glandular stomach. Control rats treated with ethanol alone had gastric lesions of 48.8" 4.3 mM ŽTable III.. SKP-450 Ž0.1]1.0 mg kgy1 . reduced mucosal lesions at lower doses Ž25.9" 3.2 and 33.5 " 1.0 mM, 46.9 and 31.4% inhibition ¨ s control, at 0.1 and 0.2 mg kgy1 , respectively, P- 0.05.. At higher doses, however, SKP-450 did not reduce the mucosal lesions in 95% ethanol-treated rats. Omeprazole Ž100 mg kgy1 , p.o.. protected significantly from the ethanol-induced gastric mucosal lesions Ž12.6" 10.4 mM, 74.1% inhibition ¨ s control.. As shown in Table IV, the mucosal damage was
more severe in 0.3 N NaOH-treated rats than in 95% ethanol-treated rats Žcontrol rats: 110.9" 15.6 mM.. The effect of SKP-450 in 0.3 N NaOH-treated rats had shown a biphasic pattern similar to those in ethanol-treated rats. SKP-450 Ž0.5]3.0 mg kgy1 . significantly reduced mucosal lesions at lower doses Ž76.0" 3.6 and 39.5" 4.2 mM, 31.5 and 64.3% inhibition ¨ s control, at 0.5 and 1.0 mg kgy1 , respectively, P- 0.05.. However, the mucosal lesions were not significantly protected upon the treatment of 3.0 mg kgy1 of SKP-450 Ž100.8" 14.9 mM, 9.1% inhibition ¨ s control.. Glibenclamide itself did not influence on the control ulcer index Ždata not shown., but it significantly inhibited the SKP-450 Ž1.0 mg kgy1 .-induced reduction of mucosal lesions Ž116.1" 13.0 mM, P- 0.01 ¨ s 1.0 mg kgy1 of SKP-450.. Omeprazole Ž100 mg kgy1 , p.o.. significantly reduced mucosal lesions Ž42.1" 17.7 mM, 62.1% inhibition ¨ s control. in NaOH-treated rats.
DISCUSSION The results of the present study indicate that SKP450 has a potent anti-ulcer activity. SKP-450 protected against the gastric lesions induced by either ethanol or NaOH. In addition, SKP-450 inhibited the basal as well as histamine-stimulated gastric acid secretion. Previous studies have shown that SKP-450 exerts vasorelaxation in rat aorta and its action may be mediated by ATP-sensitive potassium channel w2, 4x. Similarly, it was suggested that the activation of ATP-sensitive potassium channel is an important mechanism for hyperpolarisation and vasodilation in various tissues w18x. On the other hand, the activation of ATP-sensitive potassium channel could contribute to the inhibition of gastric secretion by the reduction of intracellular calcium concentration. In the present study, SKP-450 has been found to have a potent anti-secretory activity ŽED50 value for basal acid secretion: 0.12 mg kgy1 .. This effect is comparable with or more potent than that of omeprazole, a potent anti-ulcer agent clinically used.
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Table IV Effects of SKP-450 on the NaOH-induced gastric lesions Treatment
Dose (mg kg y 1)
Control SKP-450 SKP-450 SKP-450 SKP-450 Ž1 mg kgy1 . qGlibenclamide Ž20 mg kgy1 . Omeprazole Omeprazole
] 0.5 1.0 3.0
Ulcer index (mm) 110.9" 15.6 76.0" 3.6 Ž31.5.U 39.5" 4.2 Ž64.3.U 100.8" 14.9 Ž9.1.
] 30 100
116.1" 13.0†† 85.3" 21.4 Ž23.3. 42.1" 17.7 Ž62.1.U
U Data represent the mean " SEM Ž n s 5.. Values in parenthesis mean % inhibition. P - 0.05 compared with control group. ††P- 0.01 compared with 1 mg kgy1 of SKP-450.
The anti-secretory effects of SKP-450 may be mediated by increasing potassium conductance and subsequently by interfering depolarisation-induced Ca influx. Consistent with this hypothesis, the effect of SKP-450 on histamine-stimulated acid secretion was blocked by glibenclamide, a selective inhibitor of ATP-sensitive potassium channels in the present study. These results further suggest that ATP-sensitive potassium channels are present and functioning in gastric mucosal circulation, and are involved in the anti-secretory action of SKP-450. The possible anti-ulcer effects of SKP-450 were further examined in the experimentally-induced ulcer models. SKP-450 exerted the protective effects against either ethanol or NaOH-induced gastric mucosal damage at low doses Ž0.1]1.0 mg kgy1 .. The protective effect of SKP-450 appears to be more potent than that of omeprazole. Thus it might be speculated that omeprazole has an anti-secretory activity only whereas SKP-450 possesses the cytoprotective effect as well as anti-secretory effect. In addition, acid stability of SKP-450 may also account for the potency of anti-ulcer effect when compared with omeprazole. Omeprazole, indeed, has been reported to be acid labile w19x. In our experimental conditions, SKP-450 exhibited more potent activity in 95% ethanol-induced ulcer model than in 0.3 N NaOH-induced mucosal damage; its maximal inhibition was shown at doses of 0.1 and 1.0 mg kgy1 , respectively. The reason for this difference is unknown although it is possible that different pathological or pharmacological mechanisms are involved in different ulcer models. The mechanisms involved in the cytoprotection against gastric mucosal damage include mucus stimulation, bicarbonate secretion, increase in gastric mucosal resistance, decrease in gastric motility, increase in gastric blood flow by vasodilation, etc. w20x. It was well known that the activation of ATP-sensitive potassium channel increases gastric mucosal blood flow w10x. It was also documented that cromakalim, another potassium channel activator, reduced intestinal motility via the hyperpolarization of
gastrointestinal smooth muscle cells w21x. Based on the previous reports that SKP-450 induced vasorelaxation in rat aorta by activation of ATP-sensitive potassium channel w2x, it is likely that decrease in gastric motility andror increase in gastric blood flow is responsible for the cytoprotective effect of SKP450. In conclusion, the present study demonstrates that SKP-450 possesses a pronounced anti-ulcer effect, being unique with both anti-secretory and cytoprotective activities, and its effects may be mediated by the activation of ATP-sensitive potassium channels. The exact mode of action of SKP-450, however, is not known at present, but detailed mechanistic studies are in progress. Though SKP-450 has been developed as a novel antihypertensive agent, the results of the present study opens the another possible utility of SKP-450, namely as an anti-ulcer agent.
ACKNOWLEDGEMENTS This study was supported in part by a grant from The Ministry of Science and Technology, Korea.
REFERENCES 1. Lee BH, Yoo SE, Shin HS. Hemodynamic profile of SKP-450, a new potassium-channel activator. J Cardio¨ asc Pharmacol 1998; 31: 85]94. 2. Shin HS, Seo HW, Yoo SE, Lee BH. Cardiovascular pharmacology of SKP-450, a new potassium channel activator, and its major metabolites SKP-818 and SKP-310. Pharmacology 1998a; 56: 111]24. 3. Shin HS, Seo HW, Oh JH, Lee BH. Antihypertensive effects of the novel potassium channel activator SKP450 and its major metabolites in rats. Arzneim-Forsch r Drug Res 1998b; 48(II): 969]78. 4. Kwak YG, Park SK, Kang HS, Kim JS, Chae SW, Cho KP, Yoo SE, Kim D. KR-30450, a newly synthesized benzopyran derivative, activates the cardiac ATP-sensitive Kq channel. J Pharmacol Exp Ther 1995; 275: 807]12. 5. Shen WK, Tung RT, Machuda MM, Kurachi Y. Essential role of nucleotide diphosphates in
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nicorandil-mediated activation of cardiac ATP-sensitive Kq channel. A comparison with pinacidil and lemakalim. Circ Res 1991; 69: 1152]8. Cook NS. The pharmacology of potassium channels and their therapeutic potential. Trends Pharmacol Sci 1988; 9: 21]8. Hamilton TC, Weston AH. Cromakalim, nicorandil and pinacidil: novel drugs which open potassium channels in smooth muscle. Gen Pharmacol 1989; 20: 1]9. Ito S, Kajikuri J, Itoh T, Kuriyama H. Effects of lemakalim on changes in Ca2q concentration and mechanical activity induced by noradrenaline in the rabbit mesenteric artery. Br J Pharmacol 1991; 104: 227]33. Okada Y, Yanagisawa T, Yamagishi T, Taira N. Kq channel-opening action and KRN2391-induced reduction of Ca2q sensitivity of arterial smooth muscle. Arch Int Pharmacodyn 1993; 326: 33]51. Doi K, Nagao T, Kawakubo K, Ibayashi S, Aoyagi K, Yano Y, Yamamoto C, Kanamoto K, Iida M, Sadoshima S, Fujishima M. Calcitonin gene-related peptide affords gastric mucosal protection by activating potassium channel in Wistar rat. Gastroenterolology 1998; 114: 71]6. Li MKK, Tsui CP, Sung JJY, Scremin OU, Leung FW. Potassium channels participate in gastric mucosal protection in rats with partial portal vein ligation. Hepatology 1998; 27: 1530]5. Iwata F, Koo A, Itoh M, Lam K, Leung JWC, Leung FW. Functional evidence linking potassium channels and afferent nerve-mediated mucosal protection in rat stomach. Life Sci 1997; 61: 1713]20. Kortezova N, Bayguinov O, Papasova M. Effect of
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ANTI-ULCER ACTIVITY OF SKP-450, A NOVEL POTASSIUM CHANNEL ACTIVATOR, IN RATS HYAE GYEONG CHEON a , HYUN JUNG KIM a , HYE KYOUNG MO a , SUNG EUN YOO b and BYUNG HO LEE a,U a
Screening and Toxicology Research Center, and bBio-Organic Science Di¨ ision, Korea Research Institute of Chemical Technology, a100, Jang-dong, Yusong, Taejon 305-343, Korea Accepted 9 March 1999
The anti-ulcer effects of SKP-450, a new potassium channel activator, were evaluated on basal and histamine-induced gastric acid secretion, and against experimentally-induced ulcers such as ethanol-induced and NaOH-induced gastric ulcers. In the pylorus-ligated rat, SKP-450 Ž0.1]0.5 mg kgy1 . significantly decreased volume and concentration of gastric juice, and total acid output ŽED50 : 0.12 mg kgy1 .. SKP-450 Ž0.3]3.0 mg kgy1 . also inhibited histamine-induced gastric acid secretion, maximal effects being achieved at 1.0 mg kgy1 Ž37.9% inhibition.. In the 95% ethanol-treated rats, SKP-450 significantly reduced the mucosal lesions Ž46.9 and 31.4% inhibition at 0.1 and 0.2 mg kgy1 , respectively.. A significant reduction in the ulcer index by SKP-450 was also observed in 0.3 N NaOH-treated rats Ž31.5 and 64.3% inhibition at 0.5 and 1.0 mg kgy1 , respectively.. The effects of SKP-450 on histamine-induced acid secretion and on NaOH-induced ulcers were inhibited by glibenclamide Ž20 mg kgy1 , i.v.., a selective blocker of ATP-sensitive potassium channel. These results indicate that SKP-450 possesses anti-ulcer effects and its effects may be mediated by activation of ATP-sensitive potassium channels. Q 1999 Academic Press KEY
WORDS:
SKP-450, potassium channel activator, anti-ulcer.
INTRODUCTION SKP-450 ŽKR-30450, Fig. 1. is a potent, orally active coronary and peripheral vasodilator in rats and dogs w1]3x, and is under clinical trials. It was reported that vasorelaxant effects of SKP-450 on rat aorta and its antihypertensive effects in rats and dogs were inhibited by glibenclamide, a selective blocker of ATP-sensitive potassium channel, suggesting the involvement of ATP-sensitive potassium channel in the effects of this compound w1, 2x. In addition, SKP-450 antagonized the inhibitory effect of ATP on the ATP-sensitive potassium channel activity in single rat ventricular myocytes w4x, probably via a mode of action that was distinct from those of ATP-sensitive potassium channel activators } lemakalim, pinacidil and nicorandil w4, 5x. Potassium channel activators including SKP-450 act primarily to increase cellular potassium conductance by opening membrane potassium channels, and consequently result in membrane hyperpolarizaU
Corresponding author.
1043]6618r99r090243]06r$30.00r0
tion w6, 7x. The hyperpolarization induced by potassium channel activators is believed to result in an inhibition of Ca channels w6x, a reduction of Ca2q release from intracellular Ca2q stores through the inhibition of inositol-1,4,5-triphosphate production w8x, and a reduction in sensitivity of contractile proteins to Ca2q w9x. This change of calcium flux due to activating potassium channels might influence the gastric secretion. Recent studies also revealed that the activation of ATP-sensitive potassium channels increases gastric mucosal blood flow w10x, and ATPsensitive potassium channels play an important role in protecting the gastric mucosa w11]13x. Peptic ulcers appear to result from overproduction of gastric acid andror decrease in gastric mucosal protective mechanisms. Consequently, reduction of gastric acid production as well as reinforcement of gastric mucosal protection have been main approaches for peptic ulcer therapy. Based on the previous reports that SKP-450 may induce potassium channel activation w1, 2, 4x, SKP-450 was postulated to exert an anti-ulcer activity by protection of gastric mucosa andror by inhibition of gastric secretion. Accordingly, in the present study, the effects of Q 1999 Academic Press
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244
Fig. 1. Chemical structure of SKP-450 ŽKR-30450..
SKP-450 have been evaluated on the experimentally-induced ulcers elicited by ethanol or NaOH. In addition, the effect of SKP-450 on the gastric acid secretion was examined.
MATERIALS AND METHODS
Inhibition of basal acid secretion in the pylorus-ligated rat This study conformed with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health. Gastric anti-secretory activity was determined by Shay’s method with some modification w14x. Male Sprague]Dawley ŽS.D.. rats Ž6 weeks, 180]250 g, Korea Research Institute of Chemical Technology, KRICT, Taejon, Korea. were fasted for 24 h before experiments. The pylorus of animals was ligated under diethylether anaesthesia, and then SKP-450 Ž0.1]0.5 mg kgy1 . was intraduodenally administered. The animals were allowed to recover and stabilize in individual cages. At 5 h after surgery, the rats were killed by cervical dislocation. Following isolation of the stomach, gastric juice was collected, centrifuged at 5000 rpm for 10 min, and then measured for volume. An aliquot was used for determination of acid concentration by titration with 0.1 N NaOH to an endpoint of pH 7.0 using the Orion 960 autochemistry analyser ŽBoston, MA, USA.. Acid output was calculated by multiplying the volume of gastric juice with the acid concentration.
Inhibition of histamine-stimulated gastric acid secretion For the lumen-perfused rat studies, the method of Ghosh and Schild w15x was used. Male S.D. rats Ž10 weeks, 300]350 g. were fasted for 24 h and anaesthetized with urethane Ž1.2 g kgy1 . by an intraperitoneal injection. After tracheal intubation, a polyethylene tube ŽPE 50. was inserted and fixed in the forestomach through the oesophagus, and another cannula Ži.d. 2]3 mm. was inserted into the pyloric region of the stomach through duodenum. Animals were perfused with 0.9% wrv NaCl solution Ž378C.
by using an infusion pump at a flow rate of 1.5 ml miny1 . The perfusate from the duodenum tube was collected with fraction collector Žmodel 2110, BioRad, Richmond, CA, USA. at 15-min intervals, and its volume and acid concentration were determined by using an Orion 960 autochemistry analyser. Rectal temperature was monitored and maintained at 37 " 18C with heating pad and lamp. After baseline had been stabilized postoperatively for at least 20 min, SKP-450 Ž0.3]3.0 mg kgy1 . was given intraduodenally. Histamine Ž2 mg kgy1 . in 0.9% wrv NaCl was intramuscularly injected as a secretagogue 30 min after the administration of SKP-450. The perfusate was collected for 2.5 h after the administration of the compound. Percent inhibition was calculated from the comparisons of area under the curve of acid output in the presence or absence of SKP-450. In a separate experiment, the ability of glibenclamide, a blocker of ATP-sensitive potassium channels, to antagonize the effects of SKP-450 on histamine-stimulated acid secretion was evaluated in rats. Glibenclamide Ž20 mg kgy1 ., at a dose comparable to that used by other investigators in rats w16x, was intravenously injected 20 min prior to SKP-450 administration.
Effects of SKP-450 on experimental ulcer models Gastric mucosal lesions elicited by ethanol or NaOH were produced according to the method of Robert et al. w17x. Male S.D. rats Ž6 weeks, 180]250 g. were fasted for 24 h prior to the experiments. SKP-450 Ž0.1]3.0 mg kgy1 . was orally given 1 h before oral administration of either 95% ethanol Ž1 mlrrat. or 0.3 N NaOH Ž1 mlrrat.. One hour later, the animals were killed by diethylether anaesthesia, and the stomachs were isolated. After fixing the stomach in 13 ml of 1% formalin for 1 h, the greater curvature of the stomach was opened. The lengths of macroscopical lesion were measured, summed up and compared with the group treated with either 95% ethanol alone or 0.3 N NaOH alone. All the group allocations of the experimental animals were done in a randomized order and under blinded conditions. In a separate experiment, the ability of glibenclamide to antagonize the effects of SKP-450 on NaOH-induced ulcer model was evaluated in rats. Glibenclamide Ž20 mg kgy1 ., was intravenously injected 20 min prior to SKP-450 administration.
Drugs SKP-450 and omeprazole were synthesized at Bio-Organic Science Division, KRICT. Glibenclamide was purchased from Sigma Chemical Co. ŽSt. Louis, MO, USA.. Carboxy methyl cellulose ŽCMC. was obtained from Showa Chemical Co. ŽTokyo, Japan.. Absolute ethanol was obtained from Hayman Ltd. Co. ŽEssex, UK.. Formalin Ž17%. was
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anti-secretory effect in ¨ i¨ o, SKP-450 was given intraduodenally to pylorus-ligated rats, and its effect on gastric secretion was examined. As shown in Table I, pylorus ligation for 5 h caused the accumulation of volume of gastric juice Žcontrol rats: 5.9" 0.5 ml 200 gy1 ., the increases in the acidity of the gastric juice and the total acid output Žcontrol rats: 110 " 2.8 m Eq mly1 and 661 " 64.4 m Eq 5 hy1 , respectively.. SKP-450 Ž0.1]0.5 mg kgy1 . significantly decreased the volume and the acidity of gastric juice in a dose-dependent manner Ž1.2" 0.3 ml 200 gy1 and 64.8" 8.9 m Eq mly1 at 0.5 mg kgy1 , respectively.. These reductions in both the volume and the acidity of gastric juice resulted in the decrease of total acid output. The total acid output was dose-dependently inhibited by SKP-450 at all doses tested in this experiment Ž85.9" 25.6 m Eq 5 h at 0.5 mg kgy1 , 87.0% inhibition ¨ s control.. The calculated ED50 value for SKP-450 was 0.12 mg kgy1 . Omeprazole, a known anti-secretory compound, caused a significant reduction in acid output at 3.0 mg kgy1 Ž323 " 42.9 m Eq 5 hy1 , 51% inhibition ¨ s control..
obtained from Merck Co. ŽDarmstadt, Germany.. Polyethylene glycol ŽPEG. 400 and urethane were obtained from Junsei Chemical Co. ŽTokyo, Japan.. Diethylether was obtained from Oriental Chemical Industry ŽSeoul, Korea.. SKP-450 was suspended either in 20% PEG 400 Žfor intraduodenal administration. or in 0.5% CMC Žfor oral administration.. Glibenclamide was dissolved in dimethylforamide ŽDMF. and diluted with 0.9% wrv NaCl to yield a given concentration Žthe final concentration of DMF, 20%..
Statistical analysis
All values are expressed as mean " SEM. Data were analysed by unpaired Student’s t-test and oneway analysis of variance followed by Dunnett’s test for multiple comparisons ŽSigma Stat, Jandel Co., San Rafael, CA, USA.. In all comparisons, the difference was considered to be statistically significant at P- 0.05. The ED50 values Ža dose that inhibited basal acid secretion by 50%. were obtained from a linear regression of effects ¨ s log dose.
RESULTS
Inhibition of histamine-stimulated gastric acid secretion
Inhibition of basal acid secretion in the pylorus-ligated rat
The volume of gastric content from duodenum tube was always regular and time-dependent in the anaesthetized stomach lumen perfused rat models,
In order to determine whether SKP-450 has an
Table I Effect of SKP-450 on gastric secretion in pylorus-ligated rats Treatment
Control SKP-450 SKP-450 SKP-450 Omeprazole
Dose (mg kgy 1)
Volume of gastric juice (ml 200 gy 1)
Acidity of gastric juice (m Eq mly 1)
Total acid output (m Eq 5 hy 1)
] 0.1 0.2 0.5 3.0
5.9" 0.5 2.8" 0.5U 2.1" 0.4U 1.2" 0.3U 3.6" 0.5
110 " 2.8 157 " 5.8 78.5" 18.8 64.8" 8.9U 90.8" 8.1
661 " 64.4 446 " 47.9 Ž32.5.U 165 " 42.5 Ž75.0.U 85.9" 25.6 Ž87.0.U 323 " 42.9 Ž51.1.U
Each values represent the mean " SEM Ž n s 5.; values in parenthesis mean percent inhibition; U P- 0.05 compared with control group.
Table II Effects of SKP-450 on acid output in the lumen-perfused rat stomach under histamine stimulation Treatment Control Žhistamine acid phosphate, i.m.. SKP-450 SKP-450 SKP-450 SKP-450 SKP-450 Ž1.0 mg kgy1 . qGlibenclamide Ž20 mg kgy1 . Omeprazole
Dose (mg kgy 1)
Total acid output (m Eq 150 miny 1)
] 0.3 0.6 1.0 3.0
562 " 16.3 421 " 10.2 Ž25.1.U 357 " 32.7 Ž36.5.U 349 " 6.1 Ž37.9.U 478 " 33.5 Ž14.9.U
] 1.0
468 " 26.5† 232 " 181 Ž58.7.
Data represent the mean " SEM Ž n s 6.; values in parenthesis mean percent inhibition; U P- 0.05 compared with control group; †P- 0.01 compared with 1 mg kgy1 of SKP-450.
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Table III Effects of SKP-450 on the ethanol-induced gastric lesions Treatment Control SKP-450 SKP-450 SKP-450 SKP-450 Omeprazole Omeprazole
Dose (mg kg y 1)
Ulcer index (mm)
] 0.1 0.2 0.3 1.0 30 100
48.8" 4.3 25.9" 3.2 Ž46.9.U 33.5" 1.0 Ž31.4.U 57.1" 2.1 Žy17.0. 57.9" 6.2 Žy18.6. 44.2" 12.3 Ž0.9. 12.6" 10.4 Ž74.1.U
Data represent the mean " SEM Ž n s 5.. Values in U parenthesis mean % inhibition. P - 0.05 compared with control group.
Fig. 2. Effects of SKP-450 on acid output in the lumenperfused rat stomach under histamine stimulation. Vehicle Ž`., 0.3 Žv., 0.6 Ž^., 1.0 Ž'. and 3.0 mg kgy1 ŽI. of SKP-450. Data represent the mean " SEM Ž n s 6.. U P 0.05 compared with control group.
and the administration of histamine Ž2 mg kgy1 , i.m.. in the rats had shown a reproducible acid-output profile to time ŽFig. 2.. The total acid output in control rats was 562 " 16.3 m Eq over a 150-min time period. SKP-450 Ž0.3]3.0 mg kgy1 . inhibited the total acid output in a biphasic manner ŽTable II.. SKP-450 reached its maximal effect at 1.0 mg kgy1 Ž349 " 6.1 m Eq, 37.9% inhibition ¨ s control. and its effect was reduced at the higher dose used Ž478 " 33.5 m Eq at 3.0 mg kgy1 , 14.9% inhibition ¨ s control.. Glibenclamide Ž20 mg kgy1 , i.v.. significantly blocked the inhibitory effect of SKP-450 on histamine-stimulated acid secretion Ž468 " 26.5 m Eq, P- 0.01 ¨ s 1.0 mg kgy1 of SKP-450.. For the reference, omeprazole administration Ž1.0 mg kgy1 . resulted in the considerable inhibition of total acid output Ž232 " 181 m Eq, 58.7% inhibition ¨ s control..
Effects of SKP-450 on experimental ulcer models Oral administration of 95% ethanol or 0.3 N NaOH produced severe band-like mucosal haemorrhage in the glandular stomach. Control rats treated with ethanol alone had gastric lesions of 48.8" 4.3 mM ŽTable III.. SKP-450 Ž0.1]1.0 mg kgy1 . reduced mucosal lesions at lower doses Ž25.9" 3.2 and 33.5 " 1.0 mM, 46.9 and 31.4% inhibition ¨ s control, at 0.1 and 0.2 mg kgy1 , respectively, P- 0.05.. At higher doses, however, SKP-450 did not reduce the mucosal lesions in 95% ethanol-treated rats. Omeprazole Ž100 mg kgy1 , p.o.. protected significantly from the ethanol-induced gastric mucosal lesions Ž12.6" 10.4 mM, 74.1% inhibition ¨ s control.. As shown in Table IV, the mucosal damage was
more severe in 0.3 N NaOH-treated rats than in 95% ethanol-treated rats Žcontrol rats: 110.9" 15.6 mM.. The effect of SKP-450 in 0.3 N NaOH-treated rats had shown a biphasic pattern similar to those in ethanol-treated rats. SKP-450 Ž0.5]3.0 mg kgy1 . significantly reduced mucosal lesions at lower doses Ž76.0" 3.6 and 39.5" 4.2 mM, 31.5 and 64.3% inhibition ¨ s control, at 0.5 and 1.0 mg kgy1 , respectively, P- 0.05.. However, the mucosal lesions were not significantly protected upon the treatment of 3.0 mg kgy1 of SKP-450 Ž100.8" 14.9 mM, 9.1% inhibition ¨ s control.. Glibenclamide itself did not influence on the control ulcer index Ždata not shown., but it significantly inhibited the SKP-450 Ž1.0 mg kgy1 .-induced reduction of mucosal lesions Ž116.1" 13.0 mM, P- 0.01 ¨ s 1.0 mg kgy1 of SKP-450.. Omeprazole Ž100 mg kgy1 , p.o.. significantly reduced mucosal lesions Ž42.1" 17.7 mM, 62.1% inhibition ¨ s control. in NaOH-treated rats.
DISCUSSION The results of the present study indicate that SKP450 has a potent anti-ulcer activity. SKP-450 protected against the gastric lesions induced by either ethanol or NaOH. In addition, SKP-450 inhibited the basal as well as histamine-stimulated gastric acid secretion. Previous studies have shown that SKP-450 exerts vasorelaxation in rat aorta and its action may be mediated by ATP-sensitive potassium channel w2, 4x. Similarly, it was suggested that the activation of ATP-sensitive potassium channel is an important mechanism for hyperpolarisation and vasodilation in various tissues w18x. On the other hand, the activation of ATP-sensitive potassium channel could contribute to the inhibition of gastric secretion by the reduction of intracellular calcium concentration. In the present study, SKP-450 has been found to have a potent anti-secretory activity ŽED50 value for basal acid secretion: 0.12 mg kgy1 .. This effect is comparable with or more potent than that of omeprazole, a potent anti-ulcer agent clinically used.
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Table IV Effects of SKP-450 on the NaOH-induced gastric lesions Treatment
Dose (mg kg y 1)
Control SKP-450 SKP-450 SKP-450 SKP-450 Ž1 mg kgy1 . qGlibenclamide Ž20 mg kgy1 . Omeprazole Omeprazole
] 0.5 1.0 3.0
Ulcer index (mm) 110.9" 15.6 76.0" 3.6 Ž31.5.U 39.5" 4.2 Ž64.3.U 100.8" 14.9 Ž9.1.
] 30 100
116.1" 13.0†† 85.3" 21.4 Ž23.3. 42.1" 17.7 Ž62.1.U
U Data represent the mean " SEM Ž n s 5.. Values in parenthesis mean % inhibition. P - 0.05 compared with control group. ††P- 0.01 compared with 1 mg kgy1 of SKP-450.
The anti-secretory effects of SKP-450 may be mediated by increasing potassium conductance and subsequently by interfering depolarisation-induced Ca influx. Consistent with this hypothesis, the effect of SKP-450 on histamine-stimulated acid secretion was blocked by glibenclamide, a selective inhibitor of ATP-sensitive potassium channels in the present study. These results further suggest that ATP-sensitive potassium channels are present and functioning in gastric mucosal circulation, and are involved in the anti-secretory action of SKP-450. The possible anti-ulcer effects of SKP-450 were further examined in the experimentally-induced ulcer models. SKP-450 exerted the protective effects against either ethanol or NaOH-induced gastric mucosal damage at low doses Ž0.1]1.0 mg kgy1 .. The protective effect of SKP-450 appears to be more potent than that of omeprazole. Thus it might be speculated that omeprazole has an anti-secretory activity only whereas SKP-450 possesses the cytoprotective effect as well as anti-secretory effect. In addition, acid stability of SKP-450 may also account for the potency of anti-ulcer effect when compared with omeprazole. Omeprazole, indeed, has been reported to be acid labile w19x. In our experimental conditions, SKP-450 exhibited more potent activity in 95% ethanol-induced ulcer model than in 0.3 N NaOH-induced mucosal damage; its maximal inhibition was shown at doses of 0.1 and 1.0 mg kgy1 , respectively. The reason for this difference is unknown although it is possible that different pathological or pharmacological mechanisms are involved in different ulcer models. The mechanisms involved in the cytoprotection against gastric mucosal damage include mucus stimulation, bicarbonate secretion, increase in gastric mucosal resistance, decrease in gastric motility, increase in gastric blood flow by vasodilation, etc. w20x. It was well known that the activation of ATP-sensitive potassium channel increases gastric mucosal blood flow w10x. It was also documented that cromakalim, another potassium channel activator, reduced intestinal motility via the hyperpolarization of
gastrointestinal smooth muscle cells w21x. Based on the previous reports that SKP-450 induced vasorelaxation in rat aorta by activation of ATP-sensitive potassium channel w2x, it is likely that decrease in gastric motility andror increase in gastric blood flow is responsible for the cytoprotective effect of SKP450. In conclusion, the present study demonstrates that SKP-450 possesses a pronounced anti-ulcer effect, being unique with both anti-secretory and cytoprotective activities, and its effects may be mediated by the activation of ATP-sensitive potassium channels. The exact mode of action of SKP-450, however, is not known at present, but detailed mechanistic studies are in progress. Though SKP-450 has been developed as a novel antihypertensive agent, the results of the present study opens the another possible utility of SKP-450, namely as an anti-ulcer agent.
ACKNOWLEDGEMENTS This study was supported in part by a grant from The Ministry of Science and Technology, Korea.
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