Effects of the new potassium channel opener JTV-506 on coronary vessels in vitro and in vivo

The vascular effects of JTV-506 ((-)-(3S,4R)-2.2-bis(methoxymethyl)- 4-[(1,6-dihydro-l-methyl-6-oxo-3-pyridazinyl)amino]-3-hydroxychroman+ ++-6- carbonitrile hemihydrate, CAS 170148-29-5), a new potassium channel opener, was evaluated in isolated coronary arteries and anesthetized dogs. JTV-506 (1 nmol/l-3 mumol/l) produced a concentration-dependent relaxation in porcine isolated epicardial large coronary arteries precontracted with KCl (30 mmol/l), phenylephrine (3 mumol/l), histamine (3 mumol/l), serotonin (5-HT; 300 nmol/l), prostaglandin F2 alpha (PGF2 alpha; 10 mumol/l), U-46619 (100 nmol/l), endothelin-1 (ET-1; 30 nmol/l) and Bay K-8644 (100 nmol/l). JTV-506 was 2.5-8.5 and 13.3-81.5 times more potent than levcromakalim (CAS 94535-50-9) and nicorandil (CAS 65141-46-0), respectively, but was less potent than nifedipine (CAS 21829-25-4). JTV-506 and levcromakalim produced almost a complete relaxation in arteries precontracted with various kinds of vasoconstrictor, except for KCl. In contrast, nifedipine produced about 80-90% relaxation in arteries, precontracted with PGF2 alpha, U-46619 and ET-1. Thus, this potassium channel opener can be characterized as an agonist-nonselective vasorelaxant. The relaxing effects of JTV-506 and levcromakalim on coronary arteries precontracted with 30 mmol/l KCl was competitively antagonized by 3 mumol/l glibenclamide, an ATP-sensitive potassium (KATP) channel blocker. In canine isolated epicardial large coronary arteries, 10 mumol/l JTV-506, 10 mumol/l levcromakalim, 100 mumol/l nicorandil and 0.1 mumol/l nifedipine eliminated 10 mmol/l 3,4-diaminopyridine-induced rhythmic contractions. In anesthetized dogs, when administered directly into the coronary artery, JTV-506 induced dose-dependent increases in coronary arterial diameter and coronary blood flow. These results suggest that JTV-506 elicits coronary vasorelaxation through activation of the KATP channel. It is expected that JTV-506 might be useful in the treatment of coronary vasospasm in angina pectoris.     

Negative inotropic properties of isradipine, nifedipine, diltiazem, and verapamil in diseased human myocardial tissue.

We investigated the inotropic responses to Ca2+ antagonists using electrically driven human papillary muscle strips and human auricular trabeculae. Specimens were obtained during cardiac surgery for mitral valve replacement [New York Heart Association (NYHA) Class II-III] or heart transplantation (NYHA IV) and during aortocoronary bypass operations. The inotropic effects were studied with cumulative concentration-response curves. All Ca2+ antagonists tested significantly (p less than 0.05) depressed force of contraction at concentrations above 0.01 mumol/L, but their potencies were different. A 50% reduction of the initial force of contraction occurred at the following concentrations (NYHA II-III): nifedipine (mean IC50) 0.09 mumol/L isradipine 0.12 mumol/L, diltiazem 0.69 mumol/L, and verapamil 0.79 mumol/L. There were no significant differences in the negative inotropic effects of any tested Ca2+ antagonist between NYHA II-III and NYHA IV. When the initial force of contraction was reduced by 90%, addition of Ca2+ increased force of contraction significantly less after diltiazem (2.76 +/- 0.4 mN), isradipine (1.82 +/- 0.23 mN), and nifedipine (1.68 +/- 0.25 mN) compared to control (4.63 +/- 0.56 mN) (NYHA II-III). The negative inotropic potencies of nifedipine and verapamil were significantly greater in human auricular trabeculae compared to papillary muscle strips (p less than 0.05). However, on the relation between therapeutic vasoactive plasma concentrations and IC25 values, an entirely different rank order of potential negative inotropism could be observed: verapamil greater than nifedipine greater than diltiazem greater than isradipine.     

Cardiac action of KB-944, a new calcium antagonist

A comparative study was made between the cardiac action of diethyl 4-(benzothiazol-2-yl)benzylphosphonate (KB-944) and those of nifedipine, diltiazem and verapamil in anesthetized dogs and isolated right atria of guinea pigs. In anesthetized dogs, KB-944 caused a dose-dependent fall in mean blood pressure and dose-dependent increase in coronary sinus outflow. Coronary sinus outflow oxygen pressure was increased with increasing coronary sinus outflow; and consequently, the coronary arterio-venous oxygen difference was decreased. The cardiac output was slightly increased, while the myocardial oxygen consumption and myocardial work were reduced. In the isolated right atria of guinea pigs, KB-944 produced a decrease in spontaneous atria rate, and at the concentration of 10(-6) g/ml atrial standstill was seen in few preparations. The contractile force was decreased by KB-944. Especially, KB-944 exerted the weakest effect on contractile force among the tested drugs. These actions of KB-944 are very similar to those of nifedipine, diltiazem and verapamil.     

Binding of [3H]-nitrendipine and [3H]-diltiazem to rat myocardial sarcolemma

The binding properties of two major and chemically distinct calcium antagonists, [3H]-nitrendipine and [3H]-diltiazem, were investigated in highly purified rat cardiac sarcolemma. In the case of [3H]-nitrendipine, there appeared a single set of high affinity binding sites with a B max of approximately 0.9 pmol X mg-1 protein and a KD of approximately 0.15 nmol/l. Because of the extremely high value obtained for KD (29 mumol/l), the specificity of [3H]-diltiazem binding seemed questionable. The specific binding of 0.1 nmol/l [3H]-nitrendipine to cardiac sarcolemma was inhibited by nitrendipine and nifedipine (1 mumol/l), only partly inhibited by verapamil (1 mumol/l), and was enhanced by diltiazem (0.1-10 mumol/l). The stimulation of [3H]-nitrendipine binding by diltiazem was associated with an increase in the number of binding sites, Bmax, but with no change in the KD or the Hill coefficient. An enantiomer of diltiazem (1-cis) neither stimulated nor inhibited the [3H]-nitrendipine binding. These results strongly suggest that major prototype calcium antagonists have distinct and variously interacting sites of action in the rat cardiac sarcolemma.     

Protection by Ca2+ channel blockers (nifedipine, diltiazem and verapamil) against the toxicity of oxidized low density lipoprotein to cultured lymphoid cells.

1. Ca2+ channel blockers from 3 different chemical classes (diltiazem, verapamil and nifedipine) were compared in their ability to inhibit low density lipoprotein (LDL) oxidation and to protect cells directly against the cytotoxicity of oxidized LDL. 2. LDL oxidation promoted either by u.v. radiations or by copper ions was inhibited by nifedipine (IC50 of 10 +/- 2 and 4 +/- 0.5 mumol l-1, respectively) whereas diltiazem and verapamil were only poorly active or completely ineffective. As expected, LDL protected from oxidation by nifedipine (nifedipine/oxidized LDL) were much less cytotoxic than (unprotected) oxidized LDL (or than LDL oxidized in the presence of diltiazem or verapamil). The cytotoxic effect correlated well with the lipid peroxidation derivatives measured as the thiobarbituric acid reactive substances (TBARS) content of LDL oxidized in the presence of Ca2+ channel blockers, which suggests that the antioxidant effect of Ca2+ channel blockers protected cells indirectly from the cytotoxic effect of oxidized LDL. 3. Nifedipine also exhibited a direct cytoprotective effect against the cytotoxicity of oxidized LDL as demonstrated by incubating cells in the presence of unprotected oxidized LDL and nifedipine (IC50 of 1 +/- 0.2 mumol l-1), whereas diltiazem and verapamil did not exhibit any significant protective effect. At the concentrations used, the protective effect of nifedipine was not due to inhibition of LDL uptake by Ca2+ channel blockers. 4. The direct protective activity of nifedipine is probably unrelated to its antioxidant properties since it did not inhibit the cellular TBARS evoked by oxidized LDL taken up by the cells, in contrast to vitamin E.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative evaluation of the vasodilator effect of verapamil, nifedipine and diltiazem on isolated perfused coronary arteries of rabbit

In experiments with isolated perfused rabbit heart, nifedipine (1, 2 and 4 micrograms) produced a dose-dependent increase in coronary outflow (P less than 0.01). On the other hand effects after verapamil and diltiazem were negligible in such doses, though in high doses (10, 20 and 40 micrograms) they produced a significant (P less than 0.01) increase in coronary outflow. In experiments where noradrenaline (0.1 micrograms/ml) was added in the perfusion fluid, nifedipine (1, 2 and 4 micrograms), as well as verapamil and diltiazem (10, 20 and 40 micrograms) produced a dose-dependent, significant increase in coronary outflow (P less than 0.01).     

Inhibition of prostaglandin E2 and superoxide anion production in rat peritoneal macrophages by the calcium antagonists nifedipine and nisoldipine.

In order to clarify the possibility of an antiatherogenic action of the calcium antagonists nifedipine (CAS 21829-25-4) and nisoldipine (CAS 63675-72-9) the effect of nifedipine and nisoldipine on phorbol myristate acetate (PMA)- and calcium ionophore A23187-stimulated O2- and PGE2 production from macrophages was investigated. Nifedipine and nisoldipine inhibited dose-dependently PMA-stimulated O2- and PGE2 production, but not A23187-stimulated PGE2 production. The 50% inhibitory concentration (IC50) of nifedipine and nisoldipine for PMA-stimulated O2- production were 60 and 8 mumol/l, respectively, whereas those for A23187-stimulated O2- were 9.3 and 2.0 mumol/l. IC50 of nifedipine and nisoldipine for PMA-stimulated PGE2 production were 3.0 and 2.8 mumol/l, respectively. The release of [1-14C]-arachidonic acid from labeled macrophages stimulated with PMA was inhibited approximately by 39 to 43% in the presence of 20 mumol/l nifedipine and nisoldipine. The increase of (Ca2+)i in macrophages induced by A23187 could not be attenuated by nifedipine and nisoldipine, and (Ca2+)i level did not alter when stimulated with PMA. These results suggest that the inhibitory mechanism of nifedipine and nisoldipine for O2- production from the macrophages appears to directly inhibit the enzyme system of the NADPH-oxidase complex through the activation of protein kinase C, and that the inhibition of PMA-stimulated PGE2 production may be due to a decrease of phospholipase A2 through protein kinase C. On the basis of the inhibitory action on O2- and PGE2 production from the macrophages, a possible mechanism of antiatherogenic effect of calcium antagonists was discussed.     

Amlodipine,but not verapamil or nifedipine,dilates rabbit femoral artery largely through a nitric oxide- and kinin-dependent mechanism

1. We investigated the nitric oxide (NO) dependence of vasorelaxation in response to different calcium channel blockers (CCB), in rabbit femoral artery in vivo. 2. Anaesthetized rabbits underwent femoral artery ligation, and blood from the proximal artery was returned distal to the ligature through a constant infusion pump. The effects of local injection of CCB on perfusion pressure and plasma nitrite+nitrate (NO(x), which reflects local NO biosynthesis) concentration in this system were determined. 3. Intra-arterial verapamil, nifedipine or amlodipine 10 micromol x kg(-1) each reduced perfusion pressure. Pre-treatment with intra-arterial N(G)-nitro-L-arginine methyl ester (L-NAME, a NO synthase inhibitor) 1 micromol x kg(-1) did not affect responses to verapamil or nifedipine, but attenuated the reduction in perfusion pressure to amlodipine, from 33.2+/-2.1% to 22.5+/-1.6% (P=0.002). 4. Intra-arterial amlodipine--unlike verapamil or nifedipine--increased femoral venous NO(x), from 9.1+/-0.4 microM to 14.1+/-0.5 microM (P=0.005). 5. The bradykinin B2 receptor antagonist HOE 140, 30 mg x kg(-1), attenuated the reduction in perfusion pressure and abolished the rise in venous NO(x) concentration, following intra-arterial amlodipine. 6. Amlodipine potently inhibited serum angiotensin converting-enzyme (ACE) activity in vitro, as effectively as enalapril at similar concentrations. 7. These results suggest that the vasorelaxant effects of nifedipine and verapamil are NO-independent, whereas those of amlodipine are partly NO-dependent, in rabbit femoral artery in vivo. This effect of amlodipine occurs through B2 receptor activation, and may be related to an increase in local bradykinin through inhibition of ACE.     

Calcium entry blocking activity of dilazep in dog coronary artery

Calcium entry blocking activities of adenosine and its potentiating compounds (dipyridamole, lidoflazine and dilazep) were studied in potassium (100 mmol/l) depolarized, dog, large coronary artery strips, in comparison to nifedipine, verapamil and diltiazem. Apparent pA2 values were calculated by using concentration-response curves for calcium before and 30 min after the addition of each dilator drug. The order of potency (using both pA2 and IC50 values) for the calcium entry blocking effect was: nifedipine greater than verapamil greater than diltiazem greater than lidoflazine greater than dilazep. Dipyridamole and adenosine had negligible calcium entry blocking activities (about 10,000 times less potent than verapamil). The calcium entry blocking activity of verapamil (using pA2 values) was 39.8 times less potent than nifedipine, and 3.6, 21.4 and 97.7 times more potent than diltiazem, lidoflazine and dilazep, respectively. The maximum relaxations induced by adenosine (3.7 X 10(-4) mol/l) and dipyridamole (5 X 10(-5) mol/l) were less than 20% that of 3 X 10(-4) mol/l papaverine. However, the other test drugs caused 80-90% relaxation under similar conditions. The relaxing effect of adenosine was inhibited by 8-phenyltheophylline (adenosine receptor antagonist) and potentiated by EHNA (an adenosine deaminase inhibitor), while dilazep-induced relaxation was not affected by these drugs. These findings suggest that the calcium entry blocking effect of dilazep in dog, large coronary artery strips is not mediated through adenosine.     

Endothelin-1-induced contraction is impaired in the tail artery of renal hypertensive rats

The contraction induced by endothelin-1 (ET-1) was evaluated in tail arteries from normotensive two-kidney (2K) and hypertensive two-kidney-one-clip (2K-1C) rats. Since the maximal effect induced by ET-1 (0.1-30 or 100 nmol/l) was lower in 2K-1C (1.11 +/- 0.10 g) than in 2K (1.46 +/- 0.14 g) tail arteries, we evaluated the possible mechanisms involved in this blunted response. The sensitivity and efficacy of ET-1 were not affected by endothelium removal in either group. ET-1 failed to induce contraction of 2K and 2K-1C arteries in Ca(2+)-free medium. The contractile response induced by 10 nmol/l ET-1 was similarly inhibited by 0.1 microM nifedipine in arteries from 2K (81.6 +/- 3.3%) and 2K-1C (81.3 +/- 3.8%) rats. The effect of nifedipine was not potentiated by 10 mumol/l SK&F 96365. The cytosolic Ca2+ concentration ([Ca2+]c) was similarly increased by 30 nmol/l ET-1 in smooth muscle cells isolated from tail arteries of 2K (30.80 +/- 11.94 nmol/l) and 2K-1C (54.06 +/- 10.98 nmol/l) rats. In conclusion, the blunted contraction induced by ET-1 in 2K-1C tail arteries was not dependent on the endothelium or on decreased Ca2+ influx through channels sensitive to nifedipine or SK&F 96365. Since the increase of [Ca2+]c upon stimulation with ET-1 was similar in 2K and 2K-1C tail artery cells, probably the sensitivity to Ca2+ is decreased in 2K-1C tail arteries.     

Dihydropyridine receptors in the pregnant human uterus in vitro

The stimulatory effect of the dihydropyridine derivative, Bay K 8644, on the isolated pregnant human uterus, and its interactions with the calcium channel blockers, nifedipine, verapamil and diltiazem and with the calmodulin inhibitor trifluoperazine were investigated. In uterine preparations showing spontaneous activity, Bay K 8644 (1 nmol/l-1 mumol/l) produced an increase in the frequency of contractions without effects on their amplitude. However, strong phasic contractions were induced in quiescent preparations. The stimulatory action of Bay K 8644 proved to be insensitive to calcium withdrawal, but was completely prevented in the presence of 1 mmol/l EGTA. Bay K 8644 shifted the inhibitory concentration-response curve of verapamil and nifedipine to the right, leaving the diltiazem- and trifluoperazine-induced effect virtually unchanged. Schild plot analysis revealed a competitive interaction of Bay K 8644 with nifedipine, while the interaction with verapamil was of the nonlinear type. These data demonstrated that the dihydropyridine derivative Bay K 8644 possesses calcium agonistic properties also in the isolated human uterus. Furthermore, the competitive interaction with nifedipine showed the existence of specific dihydropyridine receptors closely associated with the calcium channel.     

Different effects of calcium channel blockers on matrix metalloproteinase-2 expression in cultured rat cardiac fibroblasts

The cardiac effects of calcium channel blockers (CCBs) related to cardiac remodeling are inconsistent. Matrix metalloproteinases (MMPs) contribute to tissue remodeling. Cardiac fibroblasts play an important role in the regulation of collagen degradation by MMPs. Using gelatin zymography, Western blotting, Griess reagent, and a calcium kit-fluo 3, we investigated the effects of nifedipine, verapamil, diltiazem, and amlodipine on MMP-2 expression and further elucidate the mechanisms in cultured rat cardiac fibroblasts. Nifedipine increased and amlodipine decreased the expression of MMP-2; however, neither verapamil nor diltiazem altered MMP-2 expression. Nifedipine also increased nitrite production, and this increase was blunted by a nitric oxide (NO) synthases inhibitor (L-NAME). Nifedipine-induced MMP-2 expression was also blunted by L-NAME. An NO donor (sodium nitroprusside) induced MMP-2 expression. Data indicated that nifedipine might increase MMP-2 expression through a possible NO-dependent pathway. Amlodipine had no influence on nitrite production. The amlodipine-induced decrease of MMP-2 expression was abolished by two protein tyrosine kinase inhibitors, genistein and herbimycin A, indicating that amlodipine might decrease MMP-2 expression through a possible protein tyrosine kinase pathway. None of the four CCBs could alter the fluoscence intensity of fluo 3, indicating that the effects of CCBs on MMP-2 expression were independent of the variation in intracellular C2+ concentration. Our findings revealed that different CCBs exerted different effects on MMP-2 expression in cardiac fibroblasts.     

Tissue selectivity of the novel calcium antagonist sesamodil fumarate in isolated smooth muscles and cardiac muscles

The effects of the novel Ca2+ antagonist sesamodil fumarate (JAN), (+)-3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-[(3,4- methylenedioxy)phenoxy]ethyl]amino]propoxy]phenyl]-4-methyl-3-oxo-2H- 1,4-benzothiazine hydrogen fumarate (SD-3211), on isolated smooth muscles and cardiac muscles were investigated and compared with those of diltiazem, verapamil, nifedipine and nicardipine. Ca2+ antagonistic activity of SD-3211 (pA2 = 8.42) was more potent than that of diltiazem and verapamil, but less potent than that of nifedipine and nicardipine in isolated pig coronary artery. The inhibition of Ca2(+)-induced contraction by SD-3211 was not reversed by drug washout, whereas that by diltiazem was easily reversed by drug washout. SD-3211 produced a concentration-dependent relaxation (EC50 = 5.7 x 10(-8) mol/l) of KCl-contracted pig coronary artery. The order of potency of the various compounds correlated with their respective Ca2+ antagonistic activities. SD-3211 antagonized KCl-induced contraction, but not that induced by A23187, in the rabbit aorta. On the other hand, negative inotropic and chronotropic effects of SD-3211 on the guinea pig right atria approximated those of diltiazem and verapamil. These results suggest that SD-3211 exerts a potent and long-lasting Ca2+ antagonistic effect on isolated arteries, possessing pharmacological properties diverse from those of known Ca2+ antagonists with respect to tissue selectivity, i.e., it is more vasoselective than diltiazem and verapamil, and more cardioselective than nifedipine and nicardipine.     

Effects of benidipine and some other calcium channel blockers on the prognosis of patients with vasospastic angina. Cohort study with evaluation of the ergonovine coronary spasm induction test

BACKGROUND: It has been reported that the morbidity rate of vasospastic angina is higher in Japan compared to western countries, and its prognosis has already been reported. However, the prognosis of vasospastic angina in relation to coronary angiographic findings, prognostic risk factors and treatment has not yet been fully investigated. METHODS AND RESULTS: From January 2000 to October 2005, 1047 patients with vasospastic angina diagnosed by coronary angiography at Gifu University Hospital and related hospitals were registered in a cohort study (follow-up rate: 91.4%, median follow-up duration: 3.8 years). The presence of coronary artery stenosis, diabetes mellitus, total spasm, and age of more than 65 years had a negative prognostic impact on cardiovascular events. Patients were treated with calcium channel blockers such as diltiazem (CAS 33286-22-5, CAS 42399-41-7), amlodipine (CAS 111470-99-6), nifedipine (CAS 21829-25-4), and benidipine (CAS 91599-74-5). Among these calcium channel blockers, when patient background was matched by the propensity score in patients treated with calcium channel blockers only, the cardiovascular event rate was significantly lower in the benidipine group than in the diltiazem group. CONCLUSION: The study demonstrated for the first time that total spasm is a risk factor, independent of other factors, for cardiovascular events in patients with vasospastic angina. Treatment with benidipine showed a better prognosis than that with diltiazem.     

Verapamil, diltiazem and nifedipine inhibit vascular responses to noradrenaline, acetylcholine and 5-hydroxytryptamine in human saphenous vein

Verapamil (0.02-2 microM), diltiazem (0.22-8.14 microM) and nifedipine (0.29-8.96 microM) produced concentration-dependent relaxation of human isolated saphenous vein. Based on the EC50 values of the calcium entry blockers, verapamil (relative potency = 1) was 7 and 5 times as potent as nifedipine and diltiazem, respectively, in producing relaxation of the human saphenous vein. In addition, verapamil, diltiazem and nifedipine inhibited the vascular responses (i.e. contractions) produced by noradrenaline (0.03-36 microM), acetylcholine (0.05-55 microM) and 5-hydroxytryptamine (0.02-25 microM) and to KCl (10-100 mM). It was concluded that verapamil, diltiazem and nifedipine relaxed the human isolated saphenous vein, verapamil being more potent than diltiazem or nifedipine, and modified the vascular response to vasoconstrictor agents, e.g. noradrenaline, acetylcholine, 5-hydroxytryptamine and KCl. Thus verapamil, diltiazem and nifedipine may inhibit calcium influx through both potential and receptor-operated calcium ion channels.     

Comparison of the cardiovascular effects of trans-diclofurime with different types of calcium antagonists in conscious spontaneously hypertensive rats.

1. Trans-diclofurime has been shown to be a potent group II calcium antagonist in in vitro and in vivo test systems. In contrast to the dihydropyridines, group II calcium antagonists have a reduced propensity to cause reflex tachycardia due to well-balanced inhibitory effects in smooth muscle and heart. Since effects on autonomic reflexes are more reliably assessed in conscious animals, the cardiovascular effects of trans-diclofurime have been examined and compared to those of nifedipine, verapamil and diltiazem in the conscious spontaneously hypertensive rat (SHR). 2. Each SHR had an indwelling catheter in the femoral artery to record mean arterial pressure (MAP) and heart rate (HR) and a cannula in the femoral vein for drug infusion over 1 min. 3. Nifedipine (0.1-3.0 mumol kg-1 i.v.) caused dose-related falls in MAP accompanied by dose-related increases in HR. Trans-diclofurime and verapamil (0.3-3.0 mumol kg-1 i.v.) also caused dose-related decreases in MAP, but significant tachycardia was only seen at 1.0 and 3.0 mumol kg-1. Trans-diclofurime (0.3 mumol kg-1) induced a significant fall in HR. Diltiazem (1.0-10.0 mumol kg-1 i.v.) induced dose-related falls in MAP, significant bradycardia was evident with 1.0 mumol kg-1 and tachycardia with 10 mumol kg-1. Trans-diclofurime and diltiazem induced less tachycardia than nifedipine and verapamil for equivalent falls in MAP. 4. These results suggest that trans-diclofurime is a potent antihypertensive agent in conscious SHR and, like diltiazem, the hypotensive effects are associated with less tachycardia than is usually apparent with calcium antagonists such as nifedipine or verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of moracizine and ethacizine on receptors of potential-operated calcium channels and calcium-binding proteins.

The action of two antiarrhytmic drugs, moracizine (MOR, CAS 31883-05-3) and ethacizine (ETHA, CAS 33414-33-4) on receptors of potential-operated CA-channels has been investigated. ETHA binding to verapamil receptors was more effective than that of MOR (IC50 = 0.53 +/- 0.08 mumol/l, respectively). The Hill coefficient for ETHA binding was similar to that of verapamil (0.64 +/- 0.09 and 0.60 +/- 0.10, respectively). Interaction of ETHA and MOR with dihydropyridine receptors in concentrations up to 10 mumol/l was similar that of verapamil, however, MOR was less potent. MOR and ETHA did not interact with calmodulin and troponin C at concentrations up to 100 mumol/l. The influence of MOR and ETHA on enzymes dependent on Ca-binding proteins (phosphodiesterase and actomyosin ATPase) was not observed up to 100 mumol/l. Comparison of clinical and electrophysiological data with these results allows the conclusion that ETHA exerts Ca-blocking effects by the interaction with verapamil receptors on potential-operated Ca-channels.     

Inhibitory effects of calcium channel blockers on intestinal motility in the dog

Calcium channel blockers are now widely used for the treatment of cardiovascular disorders. However, data concerning their effects on intestinal motility in vivo are still rather fragmentary. Therefore, we evaluated the effects of three prototype calcium channel blockers (nifedipine, verapamil and diltiazem) on intestinal motility in five fasting, conscious dogs fitted with electrodes and strain-gauges along the small bowel. The myoelectric data were analyzed by a recently developed and validated computer program which allows accurate monitoring of intestinal spike activity. The mechanical data were analyzed by calculating a motility index. After recording of at least two migrating motor complexes (control), an i.v. infusion of one of the following calcium channel blockers was maintained for 3 h: 0.29 or 0.87 mumol/kg per h nifedipine, 1.02 or 2.04 mumol/kg per h verapamil and 1.11 or 2.22 mumol/kg per h diltiazem. Nifedipine 0.29 mumol/kg per h significantly reduced (P less than 0.05) spike activity and motility index during phases II and III without disrupting migrating motor complex cycling. The higher dose suppressed migrating motor complex cycling and almost completely abolished both spike and mechanical activities. The two doses of verapamil had effects similar to those of the two doses of nifedipine. Both doses of diltiazem significantly reduced (P less than 0.05) spike activity and motility index during phases II and III without disrupting migrating motor complex cycling. We conclude that all the agents tested, apart from their well known cardiovascular effects, also have a profound inhibitory effect on intestinal motility in vivo, the order of potency being nifedipine greater than verapamil greater than diltiazem. The search for more selective calcium channel blockers for the treatment of intestinal motor disorders with minimal cardiovascular effects is warranted.     

Drug-drug interactions evaluated by a highly active reconstituted native human cytochrome P4503A4 and human NADPH-cytochrome P450 reductase system

Catalytic activities of native human CYP3A4-mediated reactions as well as drug interactions were directly evaluated by isolated reconstituted human CYP3A4: NADPH-cytochrome P450 reductase systems. The SDS-PAGE pure CYP3A4 and human NADPH-cytochrome P450 reductase had been incorporated into a binary vesicular phospholipid system of dilauroyl-phosphatidyl-choline and phosphatidyl-serine which had proven to achieve optimal nifedipine oxidase activity (19.6 nmol nifedipine oxidized x min(-1) x nmol CYP3A4(-1)). The IC50 values of ketoconazole (CAS 65 277-42-1) (approximately 3 micromol/l), quinidine (CAS 56-54-2) (approximately 5 micromol/l), mifepristone (CAS 84 371-65-3) (-8 micromol/l), 17alpha-ethinylestradiol (CAS 57-63-6) (approximately 17 micromol/l), cimetidine (CAS 51 481-61-9) (approximately 46 micromol/l), FK 506 (tacrolimus) (CAS 104 987-11-3) (approximately 53 micromol/l), naringenin (CAS 480-41-1) (approximately 87 micromol/l), and cyclosporine A (CAS 59 865-13-3) (approximately 90 micromol/l) indicate that all these drugs have an inhibitory effect on nifedipine (CAS 21 829-25-4) metabolism, whereas the drug quinine (CAS 130-95-0) did not elicit any significant inhibition.     

Calcium antagonistic effects and the in vitro duration of actions of KW-3049, a new 1,4-dihydropyridine derivative, in isolated canine coronary arteries

The newly-developed 1,4-dihydropyridine derivative KW-3049 was investigated for calcium antagonistic effects in isolated canine coronary arteries. KW-3049 relaxed the arteries contracted by KCI-depolarization with an IC50 of 7.4 x 10(-9) M, while the IC50 of nifedipine, verapamil and diltiazem were 9.1 x 10(-9) M, 1.7 x 10(-7) M and 3.1 x 10(-7) M, respectively. Comparison with negative inotropic activities examined in electrically-driven canine papillary muscles indicated that KW-3049 was more selective for vasorelaxing versus negative inotropic activities than nifedipine, verapamil and diltiazem. KW-3049 inhibited 45Ca-uptake induced by depolarization without affecting 45Ca-uptake in polarized arteries. Inhibitory effects of KW-3049 at 10(-9) and 10(-8) M on depolarization-induced contractions of arteries exhibited no recovery for up to 4 hr after washout of the tissues, whereas those of nifedipine, nitrendipine, verapamil and diltiazem at vasoinhibitory concentrations disappeared within 1 to 4 hr after washout. The uptake and efflux of [3H]compounds of KW-3049, nitrendipine, verapamil and diltiazem were examined. The uptake of compound after 2 hr of incubation was the highest for nitrendipine. The efflux rate of KW-3049 was 1/10 or less than those of the other compounds examined. In summary, the present results in isolated coronary arteries demonstrate that KW-3049 is a potent, vasculoselective calcium antagonist whose effects persist long even after washout of tissues presumably due to its slow dissociation rate from arteries.