UK-78,282, a novel piperidine compound that potently blocks the Kv1.3 voltage-gated potassium channel and inhibits human T cell activation.

1. UK-78,282, a novel piperidine blocker of the T lymphocyte voltage-gated K+ channel, Kv1.3, was discovered by screening a large compound file using a high-throughput 86Rb efflux assay. This compound blocks Kv1.3 with a IC50 of approximately 200 nM and 1:1 stoichiometry. A closely related compound, CP-190,325, containing a benzyl moiety in place of the benzhydryl in UK-78,282, is significantly less potent. 2 Three lines of evidence indicate that UK-78,282 inhibits Kv1.3 in a use-dependent manner by preferentially blocking and binding to the C-type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at - 50 mV enhances the channel's sensitivity to UK-78,282. Decreasing the number of inactivated channels by exposure to approximately 160 mM external K+ decreases the sensitivity to UK-78,282. Mutations that alter the rate of C-type inactivation also change the channel's sensitivity to UK-78,282 and there is a direct correlation between tau(h) and IC50 values. 3. Competition experiments suggest that UK-78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK-78,282's action on the channel. 4. UK-78,282 displays marked selectivity for Kv1.3 over several other closely related K+ channels, the only exception being the rapidly inactivating voltage-gated K+ channel, Kv1.4. 5. UK-78,282 effectively suppresses human T-lymphocyte activation.

Novel nonpeptide agents potently block the C-type inactivated conformation of Kv1.3 and suppress T cell activation.

The nonpeptide agent CP-339,818 (1-benzyl-4-pentylimino-1,4-dihydroquinoline) and two analogs (CP-393,223 and CP-394,322) that differ only with respect to the type of substituent at the N1 position, potently blocked the Kv1.3 channel in T lymphocytes. A fourth compound (CP-393,224), which has a smaller and less-lipophilic group at N1, was 100-200-fold less potent, suggesting that a large lipophilic group at this position is necessary for drug activity. CP-339,818 blocked Kv1.3 from the outside with a IC50 value of approximately 200 nM and 1:1 stoichiometry and competitively inhibited 125I-charybdotoxin from binding to the external vestibule of Kv1.3. This drug inhibited Kv1.3 in a use-dependent manner by preferentially blocking the C-type inactivated state of the channel. CP-339,818 was a significantly less potent blocker of Kv1.1, Kv1.2, Kv1.5, Kv1.6, Kv3.1-4, and Kv4.2; the only exception was Kv1.4, a cardiac and neuronal A-type K+ channel. CP-339,818 had no effect on two other T cell channels (I(CRAC) and intermediate-conductance K(Ca)) implicated in T cell mitogenesis. This drug suppresses human T cell activation, suggesting that blockade of Kv1.3 alone is sufficient to inhibit this process.

Comparison of a conscious versus anesthetized canine model in response to milrinone, hydralazine and nitroprusside.

The purpose of this study was to demonstrate, based on previous and new data, that the differences between a conscious and anesthetized canine model in the cardiovascular responses to cardiovasoactive agents were beyond their difference in the sensitivity of the compensatory mechanisms. In both conscious and anesthetized canine models, mean arterial pressure (MAP) was decreased by hydralazine (at 1-3 mg/kg and 0.3-3 mg/kg; by -26.5 +/- 4.5 and -18.8 +/- 11.7% [max. changes expressed as mean +/- SEM], respectively). MAP was also decreased by nitroprusside (both at 0.01-0.1 mg/kg, by 54.6 +/- 2.8 and -60.5 +/- 3.0%, respectively) in the conscious and anesthetized models. However, the differential MAP responses to hydralazine and nitroprusside between the two models are inconsistent with a difference in the sensitivity of the two models. Hydralazine at 1 mg/kg decreased MAP greater in the conscious than anesthetized model, whereas nitroprusside decreased MAP similarly in the two models. In conclusion, not all differential responses to hydralazine and nitroprusside between conscious and anesthetized canine models can be explained by a difference in the sensitivity of their compensatory mechanisms.

Cardiovascular and renal effects of milrinone in beta-adrenoreceptor blocked and non-blocked anaesthetized dogs.

The cardiovascular effects of the low Km cAMP phosphodiesterase inhibitor milrinone (0.01-0.3 mg/kg, i.v.) were characterized in anaesthetized dogs with or without beta-adrenoreceptor blockade (nadolol, 1 mg/kg, i.v.). Heart rate was increased by milrinone at greater than or equal to 0.1 mg/kg in non-blocked dogs (61 +/- 5 beats/min [mean +/- SEM, max. change] or 40.5 +/- 6.0%) and at greater than or equal to 0.03 mg/kg in beta-blocked dogs (33 +/- 5 beats/min or 26 +/- 4%). Mean arterial pressure was decreased at greater than or equal to 0.03 mg/kg in non-blocked dogs (-34 +/- 6mmHg or -27 +/- 5%) and at greater than or equal to 0.1 mg/kg in beta-blocked dogs (-17 +/- 4 mmHg or -15 +/- 3%). These changes were or tended to be greater in non-blocked than beta-blocked dogs. The maximum rate of rise in left ventricular pressure was increased at all doses in non-blocked (3747 +/- 388 mmHg/sec or 131 +/- 14%) and beta-blocked dogs (2517 +/- 445 mmHg/sec or 131 +/- 25%), with the absolute but not percent increase being greater in non-blocked than beta-blocked dogs. Left ventricular end diastolic pressure (LVEDP) was or tended to be reduced at greater than or equal to 0.03 mg/kg in beta-blocked dogs (-3 +/- 1 mmHg or -64 +/- 20%) and at 0.01-0.1 mg/kg in non-blocked dogs (-1.4 +/- 0.9 mmHg or -50 +/- 29%). The absolute, but not percent, decrease in LVEDP at 0.03 mg/kg was greater in beta-blocked than non-blocked dogs (-2.2 +/- 0.8 mmHg or 32 +/- 10% vs. 0.0 +/- 0.7 mmHg or 0 +/- 8%). Cardiac output (CO) was or tended to be similarly increased at 0.01-0.03 mg/kg in beta-blocked (0.2 +/- 0.1/min or 15 +/- 5%) and non-blocked dogs (1.2 +/- 0.7 1/min or 40 +/- 16%). In conclusion, beta-blockade attenuated the hypotensive and chronotropic effects, but did not eliminate the positive inotropism, the reduction in cardiac preload or the increase in CO induced by milrinone in anaesthetized dogs.

Phosphodiesterase isozyme inhibition, activation of the cAMP system, and positive inotropy mediated by milrinone in isolated guinea pig cardiac muscle.

The purpose of the present study was to examine the interrelationships among phosphodiesterase (PDE) isozyme inhibition, cAMP formation, activation of cAMP-dependent protein kinase (cAPK), and positive inotropy in isolated guinea pig cardiac muscle mediated by the cardiotonic/vasodilator agent, milrinone. Milrinone was a potent and selective inhibitor of the "low Km" cAMP PDE isozyme (peak III) isolated by diethylaminoethyl ether cellulose chromatography, with IC50 values of 0.7 microM for peak III PDE and 100 microM for peak I PDE. In isolated papillary muscles frozen at peak inotropic responses, positive and significant correlations were evident between isometric force development as a function of cAMP content (r = 0.72, p less than 0.05) or cAPK activity ratio, an index of activation of cAPK (r = 0.79, p less than 0.001), for concentrations of milrinone from 0.1-1000 microM. Similar correlations were evident in muscles frozen at peak inotropic responses for the beta-adrenoreceptor agonist isoproterenol (r = 0.96, p less than 0.001; r = 0.98, p less than 0.001, respectively), but not for ouabain or Bay K-8644. The temporal sequence of these events was also quantitated for concentrations of milrinone (100 microM) and isoproterenol (3 nM) that produced approximately a 100% increase in isometric force. Whereas early time interval of force development (30 s, 1 min, isoproterenol; 30 s milrinone) were not accompanied by significant increases in either cAMP content or cAPK activity ratio, peak increases in force development for both isoproterenol (2 min) and milrinone (1 min) were related to peak increases in cAPK activity ratios. In summary, these results show that significant increases in cAMP content or cAPK activation are correlated with positive inotropy in isolated guinea pig papillary muscles with milrinone. These correlations occur at concentrations of milrinone that inhibit cardiac PDE isozymes and are similar to the known cAMP-dependent cardiostimulant isoproterenol. These data support the hypothesis that selective PDE isozyme inhibition is a mechanism by which milrinone effects positive inotropy.

The effect of extracellular Ca2+ and related ions on the cardiac action of milrinone.

The biphasic single dose, dose-response curve of milrinone was sensitive to [Ca2+]0. At concentrations of 1.8 nM Ca2+ or less this biphasic response is observed but at [Ca2+]0 of 4.5 mM or more the dose response curve becomes monotonic. The inotropic response to milrinone in contrast to norepinephrine is highly sensitive to the extracellular [Ca2+]0. At low [Ca2+]0 of 0.15 mM milrinone could produce a negative inotropic effect. The positive inotropic effect of milrinone was proportional to [Ca2+]0 up to 2.7 mM. With [Ca2+]0 above 3.6 mM and low [Na+]0, the inotropic response to milrinone was reduced. These effects were due to increased [Ca2+]i and not due to the increase in contractile force produced by Ca2+. The positive inotropic effect of milrinone in contrast to norepinephrine is increased with an increase in [K+]0 possibly due to the depolarization produced by K+. The positive inotropic response to 10 micrograms of milrinone when [Ka+]0 = 4 mM was not significantly changed by Ca2+ channel blocking agents. In depolarized tissue (20 mM K+) the electropharmacological and contractile effects of milrinone are blocked by verapamil and ruthenium red. This suggests that under these conditions different mechanisms of Ca2+ channel activation are operative. Substitution of Sr2+ for Ca2+ increased contractile force and prolonged time to peak tension and relaxation time. Milrinone decreased time to peak tension but had no detectable effect on relaxation time. The results are discussed and it is suggested that milrinone acts on Ca2+ channels in the sarcolemma and intracellularly by increasing cyclic AMP which activates Ca2+ release and uptake from the sarcoplasmic reticulum.

Effect of milrinone (Corotrope) on the contractility of isolated dog ventricular muscle.

The bipyridine milrinone (Corotrope) is a new positive inotropic agent for treatment of congestive heart failure. The dose-response curves on electrically paced isolated dog ventricular trabeculae on contractile force were determined with single as well as cumulative dosages of milrinone. These dose-response curves differed both quantitatively and qualitatively. The "single-dose, dose-response" curve shows a flattening out in the dosage range of 1-5 micrograms/ml, which was followed by a second "single-dose, dose-response" curve between 5-50 micrograms milrinone/ml bathing fluid. The maximal response obtained at 50 micrograms/ml with the single dose-response was 1.44 +/- 0.15 g (132 +/- 9%), while with the cumulative dose-response maximum contractile change attained at 1 microgram/ml was 0.58 g (40 +/- 7%). This difference is possibly due to the tachyphylaxis produced by the first dose of milrinone which reduced the effects of the second dose. The biphasic dose-response to milrinone was converted to a monophasic one by raising [Ca2+] from 1.8-4.5 mM. A statistical analysis of the single-dose, dose-response curve was conducted by applying a third-degree polynomial fitted by least squares; the curve gave a statistically significant fit with the experimentally obtained data. This suggests that the plateau observed is not due to random variation and that the single-dose, dose-response curve consists of at least two portions. This point was further substantiated by showing that the Ca2+ channel blockers had an effect on the low dosages of milrinone (less than 2.5 micrograms/ml) but had no significant effect on the dosages above 5 micrograms/ml. Milrinone increases the rate of relaxation and decreases the time for 50 and 90% relaxation. The data suggest that milrinone may act on two different types of calcium channels.

The effect of milrinone (Win 47203) on the in vitro electropharmacological properties of mammalian cardiac tissue.

The electropharmacological effects of milrinone (Win 47203), a new positive inotropic agent of the bipyridine class, were studied on dog and guinea pig cardiac muscle by means of microelectrode techniques. In dog Purkinje fibers perfused with a Krebs solution containing 4 mM K+, milrinone did not produce any changes in action potential configuration. However, rate of discharge from spontaneously active Purkinje fibers was increased by milrinone. In dog auricular and ventricular trabeculae, milrinone increased action potential amplitude, overshoot, and phase 2 of the action potential. The duration of the action potential and the effective refractory period were decreased. In guinea pig papillary muscle, the changes in the action potential produced by milrinone consisted of a slight increase in overshoot and a reduction in the duration of the action potential. Dog Purkinje fibers depolarized with 20 mM K+ did not respond to milrinone. When such depolarized Purkinje tissue was treated with norepinephrine, slow action potentials appeared, and these could be increased by milrinone. Depolarized dog trabeculae were quiescent and addition of milrinone produced a slow action potential; the amplitude, rate of depolarization, and duration of the slow action potentials were dependent on the milrinone concentration. In depolarized guinea pig papillary muscles, milrinone induced dose-dependent slow action potentials and contractions which could be reversed on washing. Preexisting slow action potentials induced by tetraethylammonium plus high [Ca]o were potentiated by milrinone in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological actions of amrinone in dogs with cardiac lesions.

We examined the actions of amrinone in five models using dogs to determine under what circumstances intravenous amrinone might exert arrhythmogenic or antiarrhythmic properties. In dogs with 24-h post-coronary artery ligation arrhythmias, amrinone, given at incrementally increasing doses of 1.5, 3.0, and 6.0 mg/kg at 30-min intervals, produced significant increases of cardiac contractility without altering the severity of the arrhythmia. In dogs with 2- to 6-day-old ischemic lesions and 90-100% sinus beats, a bolus dose of 3.0 mg/kg amrinone was followed by an increased incidence of abnormal beats (p = 0.013); neither 1.5 nor 6.0 mg/kg caused a significant incidence of arrhythmias. Acute occlusion of the left anterior descending coronary artery followed by reperfusion caused fibrillation in nine of 15 control dogs and two of 14 dogs treated with 2.3 mg/kg amrinone. This difference was significant at the level p less than 0.05. In ouabain-intoxicated dogs, amrinone at 1.0 and 3.0 mg/kg neither worsened nor improved the arrhythmias. In the atrial circus flutter arrhythmia, amrinone increased ventricular heart rate by a significantly greater amount than it increased atrial rate, suggesting that amrinone facilitates atrioventricular conduction.

In vitro electrophysiologic properties of amrinone in mammalian cardiac tissue.

We studied the actions of amrinone on transmembrane electrical activity in isolated normal and physiologically compromised mammalian cardiac Purkinje and ventricular tissues. No arrhythmogenic effects of amrinone were identified in canine Purkinje tissue superfused with concentrations of 18.7 and 187 micrograms/ml (10(-4) and 10(-3) M) or in feline papillary tissue at concentrations of 18.7 and 56.1 micrograms/ml (10(-4) and 3 X 10(-4)M). In K+-depolarized canine Purkinje tissue, amrinone at 100 micrograms/ml (5.3 X 10(-4) M) failed to restore excitability; however, in norepinephrine-activated, K+-depolarized Purkinje tissue, amrinone significantly increased slow potential upstroke velocity and action potential amplitude and duration at doses ranging from 10 to 300 micrograms/ml. In K+-depolarized canine trabecula carneae, amrinone initiated slow potentials in the absence of norepinephrine and produced dose-dependent increases in slow potential upstroke velocity and in action potential amplitude and duration over a dose range of 30-300 micrograms/ml. Amrinone at 100 micrograms/ml did not influence transmembrane electrical activity of Purkinje tissue with tetracaine-depressed Na+-channel function. Amrinone did not produce oscillatory afterpotentials, influence ouabain-induced afterpotentials, or alter overdrive-induced depolarization. Amrinone caused no distinctly arrhythmia-producing effects in a heterogeneous population of depolarized canine Purkinje tissues removed from canine myocardial infarction, although small changes were observed in some tissues exhibiting slow channel action potentials. These experiments show that amrinone did not influence either Na+-dependent inward currents or Na+-K+ exchange mechanisms. Instead, amrinone facilitated slow channel action potentials in cardiac tissue and thereby could alter cardiac contractility, as well as conduction within reentry circuits in hearts which possess appropriate pathophysiologic substrates.